KR20030021167A - Diagnostic methods for pompe disease and other glycogen storage diseases - Google Patents

Abstract

The present invention provides methods for screening lysosomal accumulators, preferably glycogen accumulators, from a subject using tetrasaccharide as a biomarker. In a more preferred embodiment, the subjects are screened for Pompe disease (ie, glycoaccumulative disease type II). The present invention also provides neonatal screening assays. Furthermore, the present invention provides methods for monitoring clinical conditions and the effectiveness of therapeutic treatment in subjects with disease. The present invention also provides methods for measuring tetrasaccharide biomarkers by tandem mass spectrometry, preferably as part of a neonatal screening assay for Pompe disease.

Description

Diagnostic methods for pompe disease and other glycogen storage diseases

Related Application Information

This application claims priority to US Provisional Application No. 60 / 209,920, filed June 7, 2000, which is incorporated herein by reference in its entirety.

Pompe disease, also known as glycogen storage disease type II (GSD-II) or acid maltase deficiency, results from an active deficiency of the lysosomal acid α-glucosidase (GAA), a glycogen degrading enzyme It is a genetic disease of glycogen metabolism (Hirschorn, R. (1995) in The Metabolic and Molecular Bases of Inherited Disease, 7th Edition, Volume 2 (Scriver, CR, Beaudet, AL, Sly, WS, and Vale, D. Eds), pp. 2443-2464, McGraw-Hill, New York). In the most severe form, the disease is characterized by early cardiac hypertrophy, massive cardiomegaly, macroglossia, progressive muscle weakness, and marked hypotension. Most infant patients are diagnosed between three and six months of age and die before one year of age.

Recently, Chinese hamster ovary (CHO) cell lines (Van Hove JLK, et al. (1996) Proc. Natl. Acad. Sci., USA. 93 : 65-70), and transgenic mice and rabbit milk ( Bijvoet AGA, et al. (1998) Hum Mol Genet. 7 : 1815-24; Bijvoet AGA, et al. (1999) Hum Mol Genet. 8 : 2145-53), a recombinant human precursor rhGAA was prepared. rhGAA has been shown to correct defects in animal models and patient cells (Kikuchi T, et al. (1998) J Clin Invest 101, 827-833; Bijvoet AGA, et al. (1999) Hum Mol Genet. 8 : 2145- 53), gene therapy vectors have been applied to correct all defective muscles in mouse models (Amalfitano, A. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 8861-8866). Preliminary studies of human Pompe disease patients have shown that rhGAA can improve cardiac and skeletal muscle function in these patients (Amalfitano, A. et al. (2001) Genet. Med. 3: 132). These promising new therapies have led to the need for biomarker assays that are appropriate for both early diagnosis and therapeutic monitoring.

At present, there are no known biomarkers that can be easily used (and non-invasive) as a diagnosis of Pompe disease. The development of screening assays for Pompe disease is particularly helpful in infant disease forms. Early diagnosis and treatment of newborns or infants suffering from Pompe disease can improve the prognosis of these patients. Moreover, monitoring therapies can improve the efficiency of treatment and prognosis for Pompe disease patients.

Using chromatographic methods, Hallgren et al. ((1974) Eur. J. Clin. Invest. 4, 429-433) reported that Glcα1-6Glcα1-4Glcα1-elevated concentrations in the urine of 10 year old patients with Pompe disease. Glucose tetramers with structures estimated to be 4Glc (Glc ₄ ) were identified and characterized.

In addition, the urine glycoside (Glc) ₄ is a type of glycogen accumulation disease type III and VI (Lennarston, G., et al. (1976) Biomed. Mass Spectrum. 3, 51-54; Oberholzer, K. and Sewell, AC (1990) ( Clin. Chem. 36, 1381), Duchenne muscular dystrophy (Lennarston, G., et al. (1976) Biomed. Mass Spectrum. 3 , 51-54; Kikuchi T, et al. (1998) J Clin Invest 101, 827-833), acute pancreatitis (Kumlien et al., (1988) Clin. Chim. Acta 176 : 39; Kumlien et al., (1989) Int. J. Pancreatol. 4 : 139; Wang, WT, etc. (1989) Anal Biochem 182, 48-53 ), in particular malignant tumors (Kumlien, etc., (1988.) Clin Chim Acta 176:..... 39), and during pregnancy (Zopf, DA, et al. (1982 ) J. Immunol.Methods 48, 109-119; Hallgren, P., et al. (1977) J. Biol. Chem. 252, 1034-1040).

Lennarston et al . (1978) Eur. J. Biochem. At 83 : 325, urine oligosaccharides secreted from two children suffering from GSD type II or type III were characterized by gas chromatography (GC) / mass spectroscopy (MS). The major oligosaccharide secreted under these two conditions was (Glc) ₄ . Larger oligosaccharides were also present. Similarly, Chester et al (1983) reported a 4 to 60-fold increase in urine (Glc) ₄ secretion in patients suffering from GSD type II and III ( Lancet 1 : 994). The researchers also reported a modest increase in urine (Glc) ₄ in clinically normal heterozygotes. Identification and quantification of oligosaccharides was performed by radioimmunoassay and gas chromatography / mass spectrometry. It is also described in Peelen et al. , (1994) Clin. Chem. 40 : 914, and Klein et al ., (1998) Clin Chemistry 44 : 2422.

Oberholzer et al. (1990) Clin. Chem. At 36 : 1381, urine (Glc) ₄ secretion was analyzed in patients suffering from GSD using high performance liquid chromatography (HPLC). This study found that (Glc) ₄ secretion in urine is not only related to muscle symptoms but also liver symptoms in patients with GSD.

None of the studies described above evaluated the plasma concentration of (Glc) ₄ in GSD patients. Moreover, these studies do not explain whether the concentration of (Glc) ₄ rises during neonatal period when compared to healthy subjects. Furthermore, the references refer to (Glc) ₄ for diagnosing Pompe disease, evaluating the severity of the disease, or monitoring the clinical status of Pompe disease, for example in analyzing the effectiveness of therapeutic therapies. It does not suggest that it can be used for.

Various methods have been developed to analyze (Glc) ₄ , which is followed by permethylation of fractionated urine oligosaccharides followed by gas-mass spectrometry analysis (Lennartson, G., et al. (1976) Biomed. Mass Spectrom ... 3, 51-54), radioimmunoassay method (. Zopf, DA, etc. (1982) J. Immunol methods 48, 109-119), enzyme-connection immunosorbent assay (enzyme-linked immunosorbent assay) (Kumlien, J. et al. (1986) Glycoconjugate J. 3, 85-94 ), HPLC using monoclonal antibodies against (Glc) ₄ (Wang, WT, et al. (1989) Anal. Biochem. 182, 48-53) and HPLC methods including analysis of perbenzoylated oligosaccharides (Oberholzer, K. and Sewell, AC (1990) Clin. Chem. 36, 1381), or pulsed amperometric detection or post column derivatization (anion-exchange method by post column derivatization) (Peelen, GOH, et al. (1994) Clin. Chem. 40, 914-921). As far as the inventors of the present invention recognize, detection and quantification of (Glc) ₄ using tandem mass spectrometry has not been reported previously. Moreover, plasma concentrations of (Glc) ₄ in Pompe disease patients have not been previously reported. Furthermore, protocols using (Glc) ₄ as a biomarker for Pompe disease during neonatal period have not been previously proposed.

Meikle et al. (1997) Clin. Chem. 43 : 1325 and WO 97/44668 describe the use of the lysosomal membrane protein LAMP-1 as a general diagnostic marker for lysosomal accumulators. LAMP-1 concentrations were measured in healthy subjects as well as subjects suffering from one of 25 lysosomal accumulators using time-resolved fluorescence immunoassay in plasma samples. It became. LAMP-1 was elevated in plasma samples of subjects suffering from 17 of the 25 diseases evaluated. However, although all four subjects in the screened four subjects with Pompe disease had severe clinical syndromes, only one of them showed an elevated plasma LAMP-1 concentration. LAMP-1 and lysosomal enzyme activities have also been characterized in fibroblast cell lines established from patients with Pompe disease.

Hua et al. (1998) Clin. In Chemistry 44: 2094, a second lysosomal membrane protein, LAMP-2, was used as a biomarker for screening lysosomal accumulators. LAMP-2 was measured in plasma from healthy subjects and diseased subjects using fluorescence-immunoquantification. Plasma levels of LAMP-2 have been shown to be elevated in subjects with 14 of the 25 lysosomal accumulators evaluated. However, none of the four subjects suffering from Pompe disease showed an elevation of LAMP-2. Concentrations of LAMP-1 and LAMP-2 were also measured in neonatal blood spots from a "unpartitioned" newborn population. LAMP-1 and LAMP-2 concentrations in newborns were elevated compared to levels in older subjects. This report suggests that primary screening by such lysosomal membrane biomarkers may cause a high rate of false positives. The researchers suggest further testing with second-tier diagnostic methods for 1 to 5% of the neonatal population.

Thus, there is a need in the art for a method of identifying subjects suffering from Pompe disease, especially during neonatal period. There is also a need in the art for non-invasive methods of identifying and monitoring individuals suffering from Pompe disease. There is also a need in the art for neonatal screening methods for Pompe disease, compatible with existing methods for screening other hereditary metabolic diseases.

The present invention relates to a method for diagnosing and monitoring subjects suffering from lysosomal storage diseases, specifically, the present invention is based on the presence of biomarkers in biological fluids and tissues. And methods for diagnosing and monitoring Pompe disease (ie, Glycogen storage disease type II) and other glycogen storage diseases.

1 shows the HPLC separation chromatogram for BAB-labeled (Glc) ₄ in urine samples. The Y axis is the ultraviolet absorbance at 304 nm. The X axis shows the elution time in minutes. IS is an internal standard, 10 mg / mL of celloptose (C5). Panel A is the dissolution profile of urine obtained from normal individuals. Panel B is the dissolution profile of urine obtained from GSD type II patients.

FIG. 2 shows the HPLC separation chromatogram for BAB-labeled (Glc) ₄ in plasma samples. The Y axis is the ultraviolet absorbance at 304 nm. The X axis shows the elution time in minutes. IS is an internal standard, celloptose (C5) at 1 mg / mL. Panel A is the dissolution profile of urine obtained from normal individuals. Panel B is the dissolution profile of urine obtained from GSD type II patients.

FIG. 3 shows PMP-labeled oligosaccharides, maltohexose (M ₆ ), maltopentose (M ₅ ), maltotetraose (M ₄ ), (Glc) ₄ , maltotriose (M ₃ ), maltose (Mlt ), And HPLC analysis chromatogram of glucose (Glc). The Y axis is the ultraviolet absorbance at 304 nm. The X axis shows the elution time in minutes. Panel A is the elution profile of (Glc) ₄ and malto-oligosaccharide standards. Panel B is the dissolution profile of (Glc) ₄ in urine of type GSD II patients. Arrows indicate the absence of M ₄ .

4 shows BAB-labeled maltotetraose sodium adduct ion (M ₄ -BAB) Na +, m / z 866.4 (Panel A) ; BAB-labeled (Glc) ₄ sodium adduct ion (Glc ₄ -BAB) Na +, m / z 866.4 (Panel B) ; And product ion spectra of hexose tetramer, m / z 866.4 (Panel C) , present in GSD Type II urine samples. m / z 704.4, m / z 542.3, and m / z 509.2 product ions correspond to the deletion of one hexose, two hexoses, and BAB-labeled glucose, respectively. Y axis is the% strength of the fragments. X axis is m / z value.

FIG. 5 shows ESI-MS-MS spectra for BAB-labeled oligosaccharides in urine of glycogen accumulation disease type II patients. Derivative samples were injected directly into ESI-MS-MS after C18 cartridge purification. Ions were scanned by a quadrupole mass spectrometer (see text for experimental details). Y axis is the% strength of the fragments. X axis is m / z value.

6 shows the urinary (Glc) ₄ levels from Patient 1 (Panel A) , Patient 2 (Panel B) , Patient 3 (Panel C) . (Glc) ₄ levels are shown as mmol / mol creatine (Cr). The dashed line represents the major (Glc) ₄ level plus the standard deviation value in 20 normal controls (less than 1 year old). Open arrows indicate the starting point of enzyme therapy treatment. Closed arrows in dotted lines indicate the starting point for double enzyme administration. Solid closed arrows indicate the starting point of immunotherapy.

FIG. 7 shows plasma (Glc) ₄ levels from Patient 1 (Panel A) , Patient 2 (Panel B) , Patient 3 (Panel C) . (Glc) ₄ levels are expressed in mg / mL. The dashed line represents the major (Glc) ₄ level plus the standard deviation value in 20 normal controls (less than 1 year old). Open arrows indicate the starting point of enzyme therapy treatment. Closed arrows in dotted lines indicate the starting point for double enzyme administration. Solid closed arrows indicate the starting point of immunotherapy.

FIG. 8 graphically depicts BAB-derivatives of the tetrasaccharide portion of the internal standard reaction mixture separated by HPLC.

9 shows comparative results for Glc ₄ analysis in control and patient urine samples by HPLC or ESI-MS / MS.

FIG. 10 shows comparative results for Glc ₄ analysis in control and patient plasma by HPLC or ESI-MS / MS.

FIG. 11 shows comparative results of Glc ₄ analysis in liquid and spotted urine samples by ESI-MS / MS.

12 shows the estimated structure of Glc ₄ tetrasaccharide.

As described in more detail below, the present invention is directed to the accumulation (ie, elevated concentrations) of hexose tetramer biomarkers represented by (Glc) ₄ in biological samples collected from patients suffering from disease. Provided are methods for screening and monitoring the diseases characterized. (Glc) ₄ tetramers are particularly useful as biomarkers for screening and monitoring glycoaccumulative diseases such as GSD-II (Pompe disease). In a preferred embodiment, the methods of the invention can be used to screen newborns by analysis of (Glc) ₄ concentrations in dry blood spots (eg, on neonatal screening cards).

The estimated structure of hexose tetrasaccharide (Glc) ₄ is: α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc It was decided.

The present invention utilizes fast and reliable methods (e.g., HPLC or tandem mass spectrometry (TMS)) to analyze elevated levels of (Glc) ₄ biomarkers in an objective manner, Provide the ability to detect and / or monitor. The present invention is advantageous in terms of its sensitivity, repeatability, high resolution, simplicity, and low cost over conventional methods. Moreover, the neonatal screening assays disclosed herein are compatible with conventional neonatal screening methods for other hereditary metabolic diseases.

Thus, as a first aspect, the present invention comprises determining a concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from a subject, and comparing the concentration to a reference value. A method of screening accumulative diseases is provided, wherein in the second step, if (Glc) ₄ in the biological sample is detected at a concentration higher than the reference value, the subject is identified as having glycogen accumulation disease. Preferably, (Glc) ₄ tetrasaccharide is assumed to be α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc Has a structure. The glycogen accumulation bottle is preferably glycogen accumulation bottle type II (GSD-II or Pompe disease), GSD III, or GSD IV; More preferably, it is GSD-II.

In another aspect, the present invention includes determining a concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from a neonatal subject, and comparing the concentration to a baseline. A method for screening glycogen accumulation disease type II); In this second step, if (Glc) ₄ in the biological sample is detected at a higher concentration than the baseline, the neonatal subject is identified as having Pompe disease. Preferably, (Glc) ₄ represents a structure estimated as α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. Have The biological sample is preferably a blood, serum, plasma or urine sample (more preferably a dry blood, serum, plasma or urine sample).

In another aspect, the present invention provides a method for treating Pompe disease, comprising determining a concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from a subject, and comparing the concentration to a reference value. A method of monitoring the clinical condition of a subject suffering from type II); In this second step, if (Glc) ₄ in the biological sample is detected at a concentration higher than the reference value, it is an indication of the clinical condition of the subject. Preferably, the (Glc) ₄ biomarker is assumed to be α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc Has a structure. In certain embodiments, the methods of the invention are performed to evaluate the effectiveness of a therapeutic regimen in a subject.

In another aspect, the present invention includes determining a concentration of hexose tetrasaccharide (Glc) ₄ by tandem mass spectrometry in dry blood spots obtained from a newborn subject, and comparing the concentration to a reference value. Providing a method of screening Pompe disease (Glycogen accumulation disease type II) in neonatal subjects; In this second step, the detection of (Glc) ₄ in the biological sample at a concentration above the baseline confirms that the newborn subject suffers from Pompe disease. Preferably, the (Glc) ₄ biomarker is assumed to be α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc Has a structure.

(Glc) ₄ tetrasaccharide can be quantified or determined by any method known in the art, such as tandem mass spectrometry, mass spectrometry, HPLC, immunopurification methods, liquid chromatography, and the like. HPLC and tandem mass spectrometry are preferred, with tandem mass spectrometry being most preferred.

Another aspect of the invention provides a method for determining the concentration of oligosaccharides in a biological sample, comprising quantifying or determining the concentration of hexose tetrasaccharide (Glc) _{4 in} the biological sample obtained from the subject by tandem mass spectrometry. Method of quantification or determination. Preferably, (Glc) ₄ represents a structure estimated as α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. Have It is also preferred that a [U- ¹³ C] glucose labeled hexose tetramer is used as an internal standard for the TMS protocol.

The method of the present invention may also be performed using other oligosaccharides (eg, limit dextrin) that accumulate in a patient suffering from GSD-II as a biomarker.

These and other aspects of the invention are described in more detail in the detailed description below.

The present invention is based, in part, on the discovery that hexose tetramers (hereinafter, (Glc) ₄ ) can be used as biomarkers for screening methods for detecting glycoaccumulative disease type II (GSD-II). (Glc) ₄ was presumably identified as glucose tetrasaccharide.

Evidence further indicates that (Glc) ₄ oligosaccharides have an α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc structure. (Hallgren et al. (1974) Eur. J. Clin. Invest. 4: 429; see FIG. 12 ).

The present invention provides that (Glc) ₄ concentrations, in particular plasma (Glc) ₄ concentrations, can be used to monitor Pompe disease patients (eg, to evaluate the effectiveness of a treatment regimen); In patients with disease, (Glc) ₄ concentrations can have a significant correlation with the clinical course of the disease.

The terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification and the appended claims, the singular forms “a”, “an” and “the” are used to include the plural forms as well, unless the context clearly indicates otherwise.

The terms "pompe disease" and "glycogen accumulation disease type II" (ie, GSD-II) are used interchangeably herein, although "pompe disease" is more commonly used to refer to a disease form in infants. Used.

As used herein, the term (Glc) ₄ refers to a hexose tetramer ((hex) ₄ ) biomarker that accumulates in the biological fluids (eg, urine and plasma) of Pompe disease patients. (Glc) ₄ was presumably identified as glucose tetrasaccharides (eg, limit dextrin) that accumulate as a result of incomplete glycogen degradation due to a lack of GAA enzymes. The estimated structure of (Glc) ₄ is α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc (FIG. 12) .

Those skilled in the art will appreciate that the putative structure of (Glc) ₄ is the structure of a hexose tetramer, as determined by tandem mass spectrometry (TMS) of its butyl-para-aminobenzoate derivatives. It can be seen that. The identification of hexose components and their linkages cannot be determined by TMS analysis. Thus, one of ordinary skill in the art can include any combination of (Glc) ₄ hexose monomers (eg, glucose, galactose, mannose), and any possible glycosides between such monomers. It will be appreciated that they can be linked by combinations (eg 1 → 2, 1 → 3, 1 → 4, 1 → 6).

In addition to the conventional observations reported in the literature, the association between (Glc) ₄ and Pompe disease strongly suggests that glucose tetrasaccharide is an important component of (Glc) ₄ .

As used herein, the term "screening" refers to the method used to evaluate a subject for the presence of a disease characterized by accumulation of (Glc) ₄ , as described above. As long as the screening method is useful and beneficial in determining which individuals in a group or population suffer from a particular disease, the screening method need not be free of false positives or false negatives. The screening methods disclosed herein may be diagnostic and / or prognostic methods and / or used to monitor patient treatment.

As used herein, the term "diagnostic method" refers to a screening method performed to identify subjects suffering from a particular disease.

"Prognostic method" means a method used to help predict the method of a disease, at least in part. In other words, prognostic methods can be used to assess the severity of the disease. For example, the screening methods disclosed herein can be performed both for identifying individuals suffering from a disease to assess the severity of the disease, and / or for predicting the future course of the disease. Such methods may be useful in assessing the need for therapeutic treatment, what type of treatment to perform, and the like. In addition, prognostic methods can be used to obtain more information about how disease progresses for a particular subject (e.g., the likelihood that a particular patient will respond favorably to a particular medication, Or to classify or segregate patients into distinct sub-populations for the purpose of conducting clinical trials thereof, or to a subject previously diagnosed with a particular disease. Can be.

As used herein, the term "quantifying the concentration" or "determining the concentration" refers to measuring the concentration or level of analyte in a given sample. Typically, an absolute or relative value will be given as the concentration of the analyte in the sample as a result of the quantification or determination step. As described in more detail below, any suitable method known in the art can be used to quantify or determine the concentration of (Glc) ₄ in a biological sample according to the present invention. Also, as described in more detail below, methods of “quantitating” or “determining” the concentration of (Glc) ₄ encompass both quantitative and / or semi-quantitative methodologies.

A “quantitative” method is a method of assigning an absolute or relative value to the concentration of an analyte in a biological sample.

A "quasi-quantitative" method refers to a method that indicates that analyte concentration is above the threshold level, but does not impart absolute or relative values. Analytical methods commonly known as "dipstick" methods are examples of quasi-quantitative analyzes.

The following description of the invention relates to (Glc) ₄ oligosaccharides. The methods of the present invention can also be applied to using longer oligosaccharides (eg, any limit dextrin produced by incomplete glycogen degradation due to lack of GAA enzyme) to detect Pompe disease.

For example, (Glc) ₆ , (Glc) ₇ and (Glc) ₈ have been described in connection with the urine of patients with Pompe disease (Lennartson et al., (1978) Eur. J. Biochem. 83 : 325; Kumlien et al., (1989) Arch. Biochem. Biophys. 269 : 678). There are at least three (Glc) ₆ isomers with the following putative structures: α-D-Glc (1 → 6) -α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc (1 → 4) -D-Glc, α-D-Glc (1 → 4) -α-D-Glc (1 → 6)- α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -D-Glc (1 → 4) -D-Glc and

α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -D-Glc (1 → 4) -D-Glc. The estimated structure of (Glc) ₇ isomers is α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) (α-D-Glc (1 → 6))-D-Glc (1 → 4) -D-Glc-α-D-Glc (1 → 4) -D-Glc and α-D-Glc ( 1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -D -Glc-α-D-Glc (1 → 4) -D-Glc. The putative structure of (Glc) ₈ oligosaccharide is: α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. Hex ₅ oligosaccharides (or oligosaccharides) with unknown structure (s) have been detected by TMS in the urine and plasma of Pompe disease patients and controls by TMS, which appears to be limit dextrin of glycogen. Hexose oligomers with up to 11 residues were detected in the urine of Pompe disease patients by MALD-TOF MS (Matrix Assited Laser Desorption-Time Of Flight Mass Spectrometry) (Klein et al., (1998) Clin. Chem. 44 : 2422).

Those skilled in the art will appreciate that oligosaccharides having α- (1 → 6) glycoside linkages at non-reducing ends are more stable and preferred. Likewise, long oligosaccharides tend to be less stable than short oligosaccharides. The degree to which certain oligosaccharides accumulate in biological samples from healthy subjects is also a consideration when compared to Pompe disease patients. Finally, the presence of interfering substances is the basis for selecting oligosaccharides for use as live markers in accordance with the present invention.

In general, as an alternative to (Glc) ₄ , (Glc) ₅ , (Glc) ₆ , (Glc) ₇ , (Glc) ₈ and longer hexose oligosaccharides are preferred biomarkers, and particularly in neonatal subjects. Used. On the other hand, these longer oligosaccharides can be used as secondary biomarkers, for example to confirm false positives using (Glc) ₄ assays. Such longer hexose oligomers can be measured using a similar protocol for the detection of (Glc) ₄ . For example, for tandem mass spectrometry, the same derivatization and scan function can be used, but other masses (m / z) are detected.

(Glc) ₄ : Live markers for Pompe disease and other metabolic diseases.

(Glc) ₄ tetrasaccharides are derived from glycogen and represent by-products of incomplete glycogen degradation resulting from acid lysosomal α-glucosidase (GAA) deficiency in Pompe disease patients (eg, limit dextrin). Known. There is evidence suggesting that limit dextrin is formed when glycogen is released into the circulation, possibly as a result of cell lysis caused by glycogen accumulation in lysosomes. In the circulatory system, glycogen receives the α- amylase and neutral action of α1-4 glucosidase will cause the production of the limit dextrin (Ugorski, (1983) J. Exp Pathol 1:.. 27). (Glc) ₄ is found at elevated concentrations in biological fluids (eg, blood, plasma, serum, urine, saliva, amniotic fluid, etc.).

The accumulation of (Glc) ₄ tetrasaccharides in urine is also associated with other glycemic diseases and diseases such as GSD-III and GSD-IV, Duchenne muscular dystrophy, acute pancreatitis and other specific diseases. GSD-III is caused by a lack of glycogen debranching enzyme activity. GSD-IV is a group of heterogeneous diseases caused by a deficiency of the liver phosphorylase system. Deficiency may be a deficiency of liver kinase itself or a deficiency of kinase kinase.

Accordingly, the present invention provides a method for screening a disease in a subject characterized by accumulation of (Glc) ₄ (eg, elevated concentrations) in a biological sample collected from the subject. According to the invention, a biological sample is collected from piheom body, in the sample (Glc) in the presence or and non-existence is determined, the presence of the sample in (Glc) ₄ of ₄ confirm that the putative it has the disease bodies piheom Let it be.

On the other hand, preferably, the present invention may be quantitative or quasi-quantitative in nature. According to this embodiment, the biological sample is obtained from the subject and the concentration of (Glc) _{4 in} the biological sample is quantified or determined. If the level of (Glc) _{4 in} the biological sample is above the reference value (ie, the reference concentration), this presumably confirms that the subject is suffering from the disease. Typically, the reference value will be based on known concentrations of (Glc) ₄ for healthy populations and diseased populations appropriate for the screened subjects (eg, neonatal subjects are generally As compared to healthy and / or diseased neonatal populations).

For example, the subject can be compared to the corresponding, unselected population. On the other hand, the subject may be compared to the corresponding population of subjects who do not have a disease (ie, healthy) and / or the corresponding population of subjects who have a disease.

Since healthy (Glc) ₄ levels tend to decrease with age in healthy subjects, the subjects are preferably compared to an age-matched population (see Tables 4 and 5). Those skilled in the art will also understand that the level of (Glc) ₄ may be higher in patients who initially develop Pompe disease as compared to later onset forms.

The reference value can be selected according to any method known in the art. In certain embodiments, the reference value may be a predetermined value. On the other hand, the reference value may be determined during the analysis process. For example, samples from known diseased subjects and / or samples from diseased subjects can be tested simultaneously with the test samples and the baseline determined therefrom.

As another alternative method, test samples from a mixed population can be analyzed and baseline determined based on the distribution of results, for example, by using statistical methods known in the art.

Thus, the baseline represents a threshold value for identifying subjects suffering from the disease. The choice of baseline is not absolute. For example, a relatively low number may be beneficial for reducing the occurrence of false negatives, but may also increase the probability of false positives. Thus, as in other screening techniques, baseline designs include, but are not limited to, cost, benefit of early diagnosis and treatment, invasiveness of subsequent diagnostic methods for individuals with false positive results, and screening analysis methods. Can be based on a number of factors, including other factors that typically have to be taken into account.

Subjects can be presumably identified as suffering from the disease using any method known in the art. For example, if (Glc) ₄ levels are about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher compared to the appropriately matched control group Can be presumed to be afflicted with On the other hand, about 2, 3, 4, 5, 8, 10 or 20 times higher (Glc) than the average (mean or median) value for a moderately ill population. Subjects with ₄ concentrations can be presumably identified as suffering from disease.

Secondary biomarkers can optionally be used to confirm probable false positives in (Glc) ₄ assays. Examples of secondary biomarkers include long chain oligosaccharides (described above) found in the body fluids of Pompe disease patients. Other possible secondary biomarkers include LAMP-1 and LAMP-2 markers (Meikle et al., (1997) Clin. Chem. 43 : 1325 and WO 97/44668; Hua et al., (1998) Clin. Chemistry 44 : 2094).

In a preferred embodiment, the methods described above are lysosomal accumulators (eg, glycoaccumulants) or Duchenne muscular dystrophy, more preferably, glycogen accumulators (except GSD-I), Even more preferably, to screen for GSD-II (Pompe disease), GSD-III or GSD-IV. In the most preferred embodiment, the methods of the present invention can be used to screen Pompe disease (GSD-II) in a subject.

There are a number of lysosomal accumulators known in the art. Examples of lysosomal accumulators include, but are not limited to, GM1 gangliosidosis, Tay-Sachs disease, GM2 ganglioside accumulation (AB variant), Sandhoff Disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease (AD type), Faber Disease, Wolfman's disease, Hurler Syndrome (MPS III), Schieer's syndrome (MPS IS), Hurler-Scheie syndrome (MPS IH / S), Hunter ) Syndrome (MPS II), Sanfilippo A syndrome (MPS IIIA), San Filippo B syndrome (MPS IIIB), San Filippo C syndrome (MPS IIIC), San Filippo D syndrome (MPS IIID), Morquio ) A disease (MPS IVA), Morquio B disease (MPS IVB), Marotoaux-Lamy disease (MPS VI), Sly syndrome (MPS,), α-mannosidosis ), β-only Oz accumulation, fucosidosis, aspartylglucosaminuria, sialidosis (mucolipidosis I), galactosialidosis (goldberg ( Goldberg syndrome), Schindler disease, Mucolipidosis II (I-cell disease), Mucolipidosis III (pseudo-hurler polydystrophy), cystinosis, Sala (Salla) disease, infantile sialic acid storage disease, Batten disease (juvenile neronal ceroid lipofuscinosis, infantile neurofatty brown microsis, mucori Feedosis IV, and prosaposin.

Enzyme deficiencies associated with lysosomal accumulation according to the present invention include, but are not limited to, β-galactosidase, β-hexosaminidase A, β-hexoazinidase B, GM ₂ activator protein, glucocerebrosidase, arylsulfatase A, galactosylceramidase, acid sphingomyelinase, acid ceramidase (ceramidase), acid lipase, α-L-iruronidase, idouronic acid sulfatase, heparan N-sulfatase, α-N-acetylglucosaminida Acetylglucosaminidase acetyl-CoA, glucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, arylsulfatase B, β-glucuronidase, α-mannosidase, β-manno Sidase, α-L-fucosidase, N-aspartyl-β-glucosaminidase, α-neuraminidase, lysosomal protective protein, α-N-acetyl-galacto Saminidases (galactosaminidase), N-acetylglucosamine-1-phosphortransferase, cysteine transport protein, sialic acid transport protein, CNL3 gene product, palmitoyl-protein thioesterase, saposin Deficiency of A, saposin B, saposin C, or saposin D.

Numerous sugar accumulators are known and are described in Y.T. Chen & A. Burchell, Glycogen storage diseases. In: C.R. Scriver etc. (Eds.). The metabolic and Molecular Bases of Inherited Disease, 7th ed. New York: McGraw-Hill. 1995, pp. See 935-965. Examples of glycemic accumulation diseases include, but are not limited to, type Ia GSD (von Gierke disease), type Ib GSD, type Ic GSD, type Id GSD, type II GSD (Pompe disease or infantile type II). GSD), Type IIIa GSD, Type IIIb GSD, Type IV GSD, Type V GSD (McArdle disease), Type VI GSD, Type VII GSD, Glycogen synthase deficiency, Renal Fanconi ( Liver glycogen synthesis with Fanconi syndrome, phosphoglucoisomerase deficiency, muscle phosphoglycerate kinase deficiency, phosphoglycerate mutease deficiency, and lactate dehydrogenase ) Deficiency.

Enzyme deficiencies associated with glycogen accumulation diseases include, but are not limited to, glucose 6-phosphatase, lysosomal acid glucosidase, glycogen debranching enzymes, branching enzymes, muscle phospho Phosphaseylase, phosphorylase kinase, muscle phosphofructokinase, glycogen synthase, phosphoglucoisomerase, muscle phosphoglycerate kinase, phosphoglycerate mutase, or Deficiency of lactic acid dehydrogenase.

Preferably, the present invention is used to detect subjects with metabolic disorders of Pompe disease (ie GSD-II), lysosomal acid α-glucosidase (GAA) deficiency.

In another aspect, the present invention provides a method for screening Pompe disease in a subject, comprising quantifying or determining (Glc) ₄ concentration in a biological sample obtained from the subject. The (Glc) ₄ concentration in the biological sample collected from the subject is compared to a baseline (as the term is defined above). Detecting (Glc) ₄ concentrations in biological samples above this threshold, which may be a predetermined value, presumably confirms that the subject is suffering from Pompe disease.

In general, the methods disclosed herein allow for veterinary and medical applications. Thus, subjects may be humans, simians, canines, felines, equines, bovines, sheeps, goats, caprines, porcines, Rabbits (rogomorphs), rodents (rodents), birds (avians) and the like. Typically, however, subjects according to the invention will be human subjects, such as, for example, newborns (ie, between birth and about one week after birth), infant, child, adolescent or adult subjects. Of these, newborn subjects are preferred. As used herein, "neonatal" subjects include premature infants, as such term is used in the art.

Subjects may be part of a general population, such as, for example, a broad-based screening assay. On the other hand, subjects have metabolic diseases characterized by the accumulation of (Glc) ₄ as described above (eg, glycogen accumulation disease, more specifically, GSD-II) (eg, Having clinical symptoms). In another particular embodiment, subjects may have already been diagnosed as having a disease characterized by accumulation of (Glc) ₄ (eg, to monitor the patient's clinical condition or effectiveness of treatment). According to this embodiment, it is preferred that the subject has been diagnosed with glycogen accumulation disease (more specifically, GSD-II).

As used herein, the term “biological sample” is used to detect the accumulation of (Glc) _{4 in} the diseases described herein (eg, glycogen accumulation diseases such as GSD-II, GSD-III and GSD-IV). And any suitable body fluid, cells, or tissue that may be included (including cultured cells and tissues). Preferably, the biological sample is relatively non-invasive to the subject and less suitable for a wide range of screening assays (ie, methods that do not include surgical methods or biopsy). Can be obtained by In addition, the biological sample is preferably a body fluid sample. Examples of bodily fluid samples include, but are not limited to, plasma, serum, blood (including cord blood), urine, saliva, amniotic fluid, and the like. Of these, blood, plasma, serum and urine samples are more preferred.

On the other hand, a biological sample may be a cell or tissue sample, including cultured cells (eg, fibroblasts) or tissues, and a conditioned medium or effluent collected from the cells or tissues ( effusions). Examples of cells or tissues include muscle (eg, skeletal muscle, smooth muscle, heart muscle and diaphragmatic muscle), liver, skin, foreskin, umbilical cells or tissue, and the like. Of these, liver and muscle cells and tissues are preferred.

Alternatively, the biological sample may be provided on a solid medium such as, for example, filter paper, cotton swabs, cotton or the like. In certain preferred embodiments, the biological sample is a dry blood sample from a newborn subject, such as, for example, dried blood spots on a newborn screening card (ie, a "Guthrie" card). As a more preferred example, the biological sample may be a dry urine sample (eg, filter paper or lining of a diaper).

Subjects may be presumed to be suffering from certain diseases (eg, Pompe disease) by the screening methods of the invention described herein. In certain embodiments, additional, secondary diagnostic tests will be performed to confirm the diagnosis in these subjects. Typically, such secondary methodologies (eg, enzyme assays for tissue biopsies) are more costly, time consuming, and invasive than the screening methods described herein. For example, subjects with a higher (Glc) ₄ level than the reference concentration may be presumed to have Pompe disease, convinced of this diagnosis, and the subject has other diseases (eg, GSD-III May be selected to perform additional diagnostic tests to analyze whether the subject is suffering from a) or a healthy subject having a false positive result in the screening assay.

The present invention provides another utility in methods for monitoring the clinical course of a subject previously diagnosed as having a disease characterized by the accumulation of (Glc) ₄ as defined above. You can find it. We found that increased (Glc) ₄ concentrations in biological samples (especially plasma, blood and serum) from diseased subjects correlated with the clinical condition of the diseased subject. . Indeed, the concentration of (Glc) ₄ may be increased prior to exacerbation of other symptoms in a diseased subject and may be used as an early indicator of regression. Thus, (Glc) ₄ can be used as an indicator of treatment efficiency and the clinical condition of the patient.

Accordingly, the present invention further includes methods of monitoring the clinical condition of a subject having a disease characterized by accumulation of (Glc) ₄ . Preferably, the subject is a subject previously diagnosed with a glycogen accumulation disease, more preferably GSD-II. The clinical condition of the subject can be monitored to determine the effectiveness of treatment regimens, such as, for example, enzyme replacement therapy, gene therapy, and / or diet. For example, if the level of biomarker suggests that the current treatment regimen is inefficient, it may be determined to initiate another course of treatment. On the other hand, the subject's condition can be monitored to determine whether to initiate or resume treatment of the subject.

The screening methods of the present invention described herein can be any suitable method for detecting the presence or absence of (Glc) ₄ (preferably as described above) in a biological sample (preferably determining the concentration of (Glc) ₄ ). Can be performed using. Examples of such methods include, but are not limited to, chromatographic methods (eg, high performance liquid chromatography, HPLC), immunoassays (eg, immunoaffinity). Chromatography, immunoprecipitation, radioimmunoassay, immunofluorescence assay, immunocytochemical assay, immunoblotting, enzyme-linked immunosorbent assay immunosorbent assay, ELISA) and the like, liquid chromatography-mass spectrometry; Gas chromatography-mass spectrometry, TOF MS, tandem mass spectrometry (TMS) and combinations of these mass spectrometry techniques with immunopurification.

Preferred methods are simple, rapid, accurate, relatively non-invasive (eg, non-surgical), sensitive, and preferably minimize interference signals from molecules other than (Glc) ₄ . When used as a method of neonatal screening, it is more desirable that the methods are compatible with existing screening assays, and that automation and high throughput screening of samples are possible.

These methods may be wholly passive and, on the other hand, preferably, partially or wholly automated. Screening programs (eg, neonatal screening programs) for evaluating a large number of samples are generally at least partially automated to facilitate high efficiency processing of the sample. Typically, for example, data is collected and analyzed using an automated system. Biological sample (eg, blood, plasma, serum, urine, etc.) arrays or micro-arrays spotted by other preferred high efficiency treatment methods can be analyzed simultaneously. Such arrangements or microarrays may include about 10, 50, 100, 200, 300, 500, 800, 1000, 2000, 5000 or more samples.

Methods using HPLC, TOF MS, tandem mass spectrometry (TMS) are preferred, and methods using TMS are most preferred.

A preferred HPLC method for the analysis of (Glc) ₄ and other glycans in biological samples uses a C18 reversed-phase column. According to this method, the baseline separation of monomer (glucose) to heptamer (maltoheptaose) standards was carried out using a derivative of para-amino-benzoic acid (PABA) with a wavelength of 304 nm. It can be easily achieved by monitoring at.

Preferred methods of quantifying or determining (Glc) ₄ and other glycans in biological samples using TMS are described in more detail below.

For biological samples in which the concentration of the (Glc) ₄ analyte is relatively low to the limits of the detection technique, it is desirable to use a concentration step prior to the step of detecting (and, on the other hand) (Glc) ₄ in the sample. Do. As an example of an exemplary, preferred enrichment technique, immunoaffinity methods can be used to increase the concentration of (Glc) ₄ in the sample prior to the detection / quantification step. For example, immunocrystallization can be performed using an antibody that specifically recognizes (Glc) ₄ conjugated to magnetized beads. Specific anti- (Glc) ₄ antibodies are known in the art (eg, Zopf et al., (1982) J. Immunological Methods 18 : 109; Lundblad et al., (1984) J. Immunological Methods 68 : 217 Lundblad et al., (1984) J. Immunological Methods 68 : 227). Size exclusion chromatography can also be used to concentrate (Glc) ₄ in the sample.

These concentration methods can also be used to separate (Glc) ₄ analytes from contaminants or interferences.

Another aspect of the invention relates to an antibody that specifically recognizes and binds to (Glc) ₄ . The term "antibody" as used herein is meant to include all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Among them, IgM and IgG are particularly preferred. The antibodies can be monoclonal or polyclonal, can be of any species, including (eg) mouse, rat, rabbit, horse, or human, or can be chimeric antibodies. have. For example, M. Walker et al., Molec. Immunol. 26, 403-11 (1989). Antibodies are described in US Patent No. 4,474,893, or US Patent No. Recombinant monoclonal antibodies prepared according to the methods disclosed in 4,816,567. Antibodies are also described in SegAl et al., US Pat. It may also be chemically constructed by specific antibodies prepared according to the methods disclosed in 4,676,980.

Antibody fragments with specific binding sites for (Glc) ₄ can also be prepared. For example, the fragments, but not, F (ab '), which may be prepared by pepsin breakdown of the antibody molecules ₂ fragments and F (ab') disulfide (disulfide) of ₂ fragment limited to the bridge, such as those Fab fragments that can be prepared by reducing. On the other hand, Fab expression libraries may be constructed to allow for quick and easy identification of monoclonal Fab fragments with the desired specificity (WD Huse et al., Science 254, 1275-1281 (1989)).

For the preparation of antibodies, they have immunogenic properties (eg, conjugated to hapten or opsonin) to various hosts, including goats, rabbits, rats, mice, humans and others. Immunization can be achieved by injection (Glc) ₄ or a derivative thereof. Depending on the species of the host, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvants, inorganic gels such as aluminum hydroxide, and lysolecithin, pluronic polyols, polyanions ( surface active substances such as polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Among the adjuvant used in humans, bacilli Calmette-Guerin (BCG) and Corynebacterium parvum are particularly preferred.

Monoclonal antibodies against (Glc) ₄ can be prepared by using any technique to produce antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler, G. et al. (1975) Nature 256 : 495-. 497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 : 31-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80 : 2026-2030; Cole, SP et al. 1984) Mol. Cell Biol. 62 : 109-120). In brief, the process is as follows: The animal is immunized with (Glc) ₄ or an immunogenic derivative or conjugate thereof (eg conjugated to hapten or opsonin). Next, lymphocytes (e.g., splenic lymphocytes) are obtained from the immunized animal and fused with immortalized cells (e.g., myeloma or heteromyeloma). Mixed cells are prepared. Hybrid cells are screened to identify hybrid cells that produce the desired antibody.

Human hybridomas secreting human antibodies can be prepared by the techniques of Kohler and Milstein. Although human antibodies are particularly suitable for human treatment, in general, the production of stable human-human hybridomas for long-term production of human monoclonal antibodies may be difficult. Hybridoma production in rodents, especially mice, is a well known method, and thus stable murine hybridomas can provide an unlimited source of antibodies with select properties. As an alternative to human antibodies, mouse antibodies can be converted to chimeric mouse / human antibodies by genetic engineering techniques. See VT Oi et al. , Bio Techniques 4 (4) : 214-221 (1986); LK Sun et al. , Hybridoma 5 (1986).

Monoclonal antibodies specific for (Glc) ₄ can be used to prepare anti-idiotypic (antitope-specific) antibodies. See McNamara et al., Science 220 , 1325-26 (1984), RC Kennedy, et al., Science 232 , 220 (1986).

In addition, techniques developed to produce “chimeric antibodies” may be used, ie, techniques for splicing mouse antibody genes to human antibody genes to obtain molecules with adequate antigen specificity and biological activity. (SL Morrison, et al. Proc. Natl. Acad. Sci. 81 , 6851-6855 (1984); MS Neuberger et al., Nature 312 : 604-608 (1984); S. Takeda, S. et al., Nature 314 ). : 452-454 (1985)). On the other hand, the techniques described for the preparation of single chain antibodies may be employed to prepare (Glc) ₄ -specific single chain antibodies using methods known in the art. Antibodies with associated specificities, but with distinct genotyping compositions, may also be prepared by chain shuffling from random combinatorialimmunoglobulin libraries (DR Burton, Proc. Natl. Acad. Sci. 88, 11120). -3 (1991)).

Antibodies can also be prepared by inducing in vivo production in lymphocyte populations or by screening immunoglobulin libraries or panels of highly specific binding reagents (R. Orlandi et al., Proc. Natl. Acad. Sci. 86 , 3833-3837 (1989); G. Winter et al., Nature 349 , 293-299 (1991)).

Newborn screening

The methods described herein may be preferably used as part of a neonatal screening program that identifies individuals suffering from disease at neonatal period to enable early treatment. Many neonatal screening programs rely on a unique sample collection method that absorbs blood from the heel onto a neonatal screening card (eg, cotton-fiber filter paper). Phenylalanine hydroxylase deficiency causing phenylketouria (PKA), and branched-chain ketoacid dehydrogenase causing maple syrup urine disease (MSUD) Increased mortality and morbidity caused by deficiencies in amino acid metabolism such as deficiency can be avoided due to the development of simple biochemical tests for elevated amino acid levels in these dry neonatal blood spots.

The screening methods described herein further may, for example, simultaneously or in parallel with other neonatal screening assays for neonatal blood samples or dry blood spots on a neonatal screening card (ie, the same sample but need not be the same assay). It may be advantageously performed.

The neonatal screening card may be comprised of any suitable natural or synthetic material, including but not limited to cotton, cellulose, acetate, and combinations thereof.

On the other hand, the neonatal screening program may be based on measuring (Glc) ₄ in any other biological sample, as described above. For example, (Glc) ₄ may be measured in blood (eg cord blood), plasma, serum, or urine. In certain embodiments, urine may be extracted from the diaper material.

Accordingly, another aspect of the invention, a newborn in a biological sample collected from piheom body (Glc) comprises the step of quantitatively or determine the _four concentrations of, in a newborn a subject (as described above) of (Glc) ₄ A method of screening for diseases characterized by accumulation, and when (Glc) ₄ is detected above a reference concentration in a biological sample, it is confirmed that the newborn subject suffers from the disease.

A preferred method of screening for Pompe disease in newborns a subject is a newborn in a blood sample collected from piheom body (Glc), the reference value or more in a biological sample comprises the step of quantitatively or determine the _four levels of (Glc) ₄ Is detected, the neonatal subject is confirmed to have Pompe disease. Preferably, the blood sample is collected from a newborn screening card. As mentioned above, this method may identify neonatal subjects suffering from other diseases such as GSD-III and other glycemic diseases. This feature of the methods of the present invention does not impair the application of these screening methodologies and can, in fact, be considered as a beneficial advantage. On the other hand, other biological samples from newborn subjects such as, for example, urine (eg, collected from pieces of diaper material) may be used.

In another preferred embodiment, described in more detail below, methods comprising tandem mass spectrometry use GSD-II (and / or other sugars) using (Glc) ₄ as a biomarker for the presence of disease. As part of a neonatal screening program for accumulators).

As mentioned above, it is desirable that the methods of neonatal screening be at least partially automated (as described above). For example, once the sample is loaded into an HPLC column or tandem mass spectrometer, it is desirable that data be input and analyzed using an automated system.

Methods based on tandem mass spectrometry (TMS)

TMS is a preferred method for carrying out the methods of the invention described herein. The concept of TMS for the analysis of mixtures using triple quadrupole mass spectrometers derives from tandem quadrupole mass spectrometry by Yost and Enke (In: Tandem Mass Spectrometry, FW McLafferty (Ed.), Wiley & Sons, New York, (1983), pp. 175-195). To selectively detect compounds of similar structure type, a precursor ion scan function to identify molecular species that are fragmented into conventional product ions or a constant neutron loss scan to identify ions that are missing conventional fragments A constant neutral loss scan function, or multiple reaction monitoring where only selected precursor and product ions are detected is used. Adding appropriate internal standards, such as stable isotope-labeled analogs to biological substrates, prior to overhaul and analysis facilitates accurate quantification of target analytes.

Any suitable TMS method known in the art can be used, including, but not limited to, triple quadrupole mass spectrometry and hybrid mass spectrometry methods combining quadrupole and TOF MS. Ion traps and ion cyclotron resonance mass spectrometers may also be used.

TMS is particularly suitable for neonatal screening programs. The ability to quantify amino acids and acylcarnitines alone can recognize more than 20 metabolic diseases. In the past, the joint research, PKU (Chace, etc., (1993) Clin Chem 39: ... 66), MSUD (Chace , etc., (1995) Clin Chem 41: ... 62), and methionine hyperlipidemia (hypermethioninemias) (Chace Et al ., (1996) Clin. Chem. 43 : 2106), and medium-chain acyl-coA dehydrogenase deficiency (MCAD) (Chace et al., (1997) Clin. Chem. 43 : 2106) all have been found to be reliably detected by TMS at neonatal time (see Sweetman, (1996) Clin. Chem. 42 : 345). Analytes are simultaneously quantified by TMS using the interlaced scan function as the sample mixture is injected into the flowing solvent. A batch method of preparing and analyzing samples in 96-well form and interpreting the results using automated computer algorithms has been reported, demonstrating the ability to analyze up to 1000 samples per day ( Rashed et al. , (1997) Clin. Chem. 43 : 1129). Neonatal screening using TMS has been implemented in a variety of fields.

To the extent that the inventors recognize, the HPLC and TMS studies described herein are the first to report increased (Glc) ₄ concentrations in plasma (or other blood-derived) samples from subjects suffering from Pompe disease. Will. Also described herein is the first TMS protocol for screening Pompe disease using (Glc) ₄ as a live marker, in particular the first such neonatal screening program.

Accordingly, the present invention also provides a method for quantifying or determining the concentration of (Glc) ₄ in a biological sample by TMS. Oligosaccharides in the sample are derivatized by any method known in the art prior to analysis, preferably with para-aminobenzoic acid (PABA) derivatives (eg butyl-PABA) or 2-aminoacridone Can be. Fragmentation of the derivatives is investigated to determine the most specific and sensitive scan function for TMS. For example, we found that butyl-PABA derivatives of (Glc) ₄ were subjected to multiple reaction monitoring using electrosprayionization-TMS (ESI-TMS) with butyl-triple quadrupole mass spectrometers. It was found that it can be detected by observing the transition from m / z 866 to m / z 509. Those skilled in the art will appreciate that other derivatization methods can be used and appropriate scan functions can be used to detect such replacement (Glc) ₄ derivatives. Likewise, many alternative ionization methods are known in the art as an alternative to ESI (eg, Matrix Assisted Laser Desorption Ionization (MALDI)).

In certain embodiments, the analyte is concentrated prior to TMS analysis, as described above. (Glc) ₄ is preferably concentrated by immunoprecipitation with conjugated paramagnetic beads to which an antibody that specifically recognizes (Glc) ₄ is conjugated. (Glc) ₄ can then be eluted from the beads using a suitable solvent. Typically, but not necessarily, the concentration step is performed prior to derivatization. In other preferred embodiments, other analytes of interest can be immunopurified from the same sample. For example, a mixed population of such beads may be added to the sample, each of which carries antibodies specific for other analytes that are characteristic of metabolic disease. In this way, the same sample can be used to screen for various hereditary metabolic diseases.

On the other hand, (Glc) ₄ can be concentrated using methods based on size exclusion.

Moreover, a "clean-up" or pretreatment step may be used to reduce or eliminate interfering or undesirable materials. For example, if the ratio of glucose to (Glc) ₄ in the biological sample is relatively high (e.g., 2: 1 or higher), then reduce the glucose concentration in the sample prior to analysis by TMS. It is preferable to make it. The concentration of glucose in the sample can be reduced by any method known in the art. As one example, a preferred method is to remove glucose, typically by treating the sample with an enzyme prior to derivatization. For example, the sample can be treated with glucose oxidase to reduce or eliminate glucose from the biological sample. The treatment with the enzyme should preferably not decompose (Glc) ₄ tetrasaccharides, or should be in small amounts even when degraded. On the other hand, glucose can be separated from (Glc) ₄ tetrasaccharides using separation (eg chromatographic) techniques, which is generally performed following the derivatization step. To illustrate this, after the derivatization step, the derivatized glucose can be separated from the derivatized (Glc) ₄ using liquid phase chromatography (eg reverse phase).

In order to ensure that the standard is placed under the same conditions as the analyte, an internal standard is generally applied to the sample prior to manipulation. Any suitable internal standard can be used. (Glc) ₄ homologues in which one of the glucose residues is substituted by [U- ¹³ C] labeled glucose to provide a mass transfer of +6 Da (as described in the Examples) is suitable and preferred. . Internal standards are applied to the sample in known amounts. The starting amount of (Glc) ₄ in the sample can be determined using a calibration curve depending on the ratio of the signals generated by the (Glc) ₄ and the internal standard in the sample. The calibration curve plots the signal ratio (signal ratio of (Glc) ₄ to internal standard) obtained for various known, (Glc) ₄ standard concentrations using a fixed amount of internal standard. to be.

Another preferred internal standard is deuterium labeled glucose tetramer.

Preferred methods of the invention for quantifying or determining (Glc) ₄ in a biological sample include: (1) collecting a biological sample; (2) adding a known amount of a suitable, stable isotope-labeled standard; (3) optionally, concentrating (Glc) ₄ in the sample by immunocrystallization with magnetized beads and eluting from the beads using a suitable solvent; (4) derivatizing glycans, for example with butyl-PABA or 2-aminoacridone; And (5) quantifying (Glc) ₄ using TMS. Optionally, as described above, interfering glucose signals can be reduced by enzymatic treatment before step (4) or by chromatographic methods before step (5).

Preferably, [U- ¹³ C] labeled glucose tetramers are used as internal standard for TMS analysis.

The aforementioned method can be used in the preferred embodiments of the screening and monitoring methods of the present invention described above. As mentioned above, such methods are preferably partially or wholly automated.

Methods based on TMS are particularly suitable for quantifying or determining (Glc) ₄ in dry blood spots from neonatal screening cards. According to this embodiment, the method further comprises extracting oligosaccharides from the dry blood spot using a suitable solvent (eg, an aqueous solvent or an aqueous / organic mixture). On the other hand, TMS may be used to quantify or determine the presence of (Glc) ₄ in dry urine samples (eg, on filter paper or diaper material).

Thus, as a particularly preferred embodiment, the present invention provides a method of preparing a blood sample, comprising: (1) providing a blood sample, typically a blood sample in the form of dry blood spots on a newborn screening card (eg, filter paper); (2) extracting oligosaccharides from the dry blood spot using a solvent; (3) adding a known amount of a suitable stable isotope-labeled internal standard to each sample; (4) derivatizing oligosaccharides (eg with butyl-PABA); (5) analyzing (Glc) ₄ derivatives by TMS using specific scan functions; (6) quantifying or determining (Glc) ₄ in the sample by comparing the signal generated by derivatized (Glc) ₄ with the signal generated by the derivatized internal standard; And (6) presumably identifying those subjects as having Pompe disease based on a (Glc) ₄ concentration in the sample that is higher than the reference (as described above). It provides a method of screening.

The invention may optionally further comprise analyte concentration steps and glucose removal steps, as described above.

As has been described above with respect to the present invention, the present invention will be described below with reference to specific embodiments, but these embodiments are only intended to illustrate the invention, not intended to limit the invention.

Example 1

Materials and devices

α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc {(Glc) ₄ }, maltotetraose (M4), Maltopentaose (M5), Maltohexaose (M6), Maltoheptaose (M7), Cellopentaose (C5), Sodium cyanoborohydride (NaBH ₃ CN), Benzo anhydride, Butyl-4-aminobenzoic acid ( BAB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and 2'-fucosyllactose were purchased from Sigma-Aldrich (St. Louis, Mo.). 2-aminoacridone (AMAC) is produced by Molecular Probes, Inc. (Eugene, OR). Methanol, acetonitrile (HPLC grade), acetic acid and hydrochloric acid were purchased from VWR Scientific products (Atlanta, GA). All other reagents were of analytical grade and were commercially available.

All HPLC solvents were filtered (0.2 μm membrane) and degassed just before use. PMP was recrystallized from methanol before use. Sep-pak Vac C18 cartridges (100 mg) and YMC-Pack Pro C ₁₈ column (250 × 4.6 mm ID, 5 μm) were purchased from Waters (Franklin, Mass.). The HPLC system was equipped with a Waters 626 pump, a 486 tunable absorbance detector, a 717 plus autosampler, and a 600S controller (Waters, Milford, Mass.). Mass spectral analysis was performed using a Quattro-LC electrospray ionization triple quadrupole tandem mass spectrometer with a Hewlett-Packard binary pump and a Gilson 215 liquid handler. MS / MS), (Micromass Inc., Beverly, Mass.). Lyophilized recombinant TVA II used for the synthesis of internal standards was donated from Takahashi Tonozuka, Ph.D. (Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo).

Example 2

(Glc) ₄ HPLC analysis for

Sample Preparation for HPLC Analysis.

In separating samples from patients undergoing enzyme replacement therapy, plasma and 6 h urine samples were collected every two weeks before and during therapy. Both urine and plasma samples (Glc) ₄ levels were frozen at −20 ° C. before testing.

Urine samples were centrifuged and 50 μl of supernatant mixed with 10 μl of deionized water containing 10 μg of cellopentaose (C5) as internal standard. Urine standards were prepared by adding 10 μl of deionized water containing a known amount of (Glc) ₄ to a 50 μl fraction of control urine containing 10 μg of C5.

Plasma or serum (200 μl) was mixed with 1 μg internal standard (C5) and 500 μl methanol in a conical glass test tube and centrifuged at 6,000 rpm for 4 minutes to precipitate denatured proteins. The supernatant was dried under nitrogen and reconstituted in 60 μl of deionized water. Plasma standards were prepared by adding a known amount of (Glc) ₄ to a 200 μl fraction of the plasma control sample.

(Glc) ₄ Derivatization of oligosaccharides for HPLC analysis.

Oligosaccharides were derivatized with butyl-p-aminobenzoate (BAB) using the method of Poulter and Burlingame ((1990) Methods Enzymol. 193, 661-689). Derivatization reagents, freshly prepared as needed, included BAB (54 mg), NaBH ₃ CN (47 mg), acetic acid (0.11 mL), and methanol (1.76 mL). 140 μl of reagent was added to each sample prepared as described above. Sample mixtures were incubated at 80 ° C. for 45 minutes and then cooled to room temperature. 0.9 ml of 15% acetonitrile was added and the mixture was vortexed for 10 seconds. Solid phase extraction was used to remove unreacted reagents from the derivatized oligosaccharides. Samples were loaded into a pre-conditioned C ₁₈ cartridge with 1 ml methanol, then 1 ml deionized water, and washed with 1 ml of 15% v / v acetonitrile / water. Next, BAB-labeled oligosaccharides were eluted with 1 ml 30% v / v acetonitrile / water. For urine samples, the eluate was analyzed directly by HPLC. For plasma samples, the eluate was dried under nitrogen gas and reconstituted in 150 μl 30% v / v acetonitrile / water prior to analysis.

2-aminoacridone (AMAC), PMP, and perbenzoyl (PB) derivatives of oligosaccharides were prepared according to known methods (Okao, G., et al. (1996) Anal. Chem. 68, 4424 -4430; Zopf, D., and Fu, D., (1999) Anal.Biochem . 269, 113-123; Daniel, PF, et al. (1981) Carbohydr.Res. 97, 161-180).

HPLC analysis and quantification.

BAB-labeled oligosaccharides were separated at a flow rate of 0.5 ml / min on a YMC-Pack Pro C ₁₈ column, and the UV absorbance of the eluate was monitored at 304 nm. HPLC elution was carried out isocratic for 30 minutes with 30% acetonitrile and 70% 0.01 mM tetrabutylammonium chloride, adjusted to pH 4-6 with 6N HCl. Excess unreacted BAB and other impurities were washed out of the column by increasing the acetonitrile to 50% by 32 minutes and returning to the initial conditions by 38 minutes. Total analysis time was 45 minutes per sample. Peak areas of (Glc) ₄ and internal standard (C5) were automatically calculated by baseline correction where appropriate and the ratio of these areas was used to quantify (Glc) ₄ .

ESI-MS / MS analysis of standards and HPLC fractions.

BAB oligosaccharide derivatives, collected in whole samples or as fractions after HPLC separation, were analyzed by ESI-MS and ESI-MS / MS on tandem mass spectrometers. Sample injection was carried out through a 20 μl Rheodyn loop into a carrier solvent of acetonitrile: water (1: 1; v / v) at a flow rate of 15 μl / min. The capillary and cone settings were 3.50 kV and 78-95 V, respectively, and the source block and desolvation temperatures were 80 and 150 ° C, respectively. Collision-induced dissociation experiments were used for collision energy 45-77 eV and gas cell pressure 3.5 × 10 ⁻³ mBar. Mass spectra were acquired in positive ion mode at a scan rate of 100 amu / s.

Example 3

By HPLC (Glc) ₄ Separation and analysis

Four derivatives were compared for suitability for quantifying oligosaccharides by HPLC using the same high-resolution liquid chromatography column, as described in Example 2 above. They were butyl-p-aminobenzoate (BAB), 2-aminoacridone (AMAC), 1-phenyl-3-methyl-5-pyrazolone (PMP), and benzo anhydride. BAB derivatization was ultimately chosen for this application based on its sensitivity, reproducibility, high resolution, simplicity and low cost advantages. In this comparative experiment, AMAC derivatization had very high sensitivity, but was unable to separate (Glc) ₄ from lactose found in large quantities in urine, and PMP derivatization had good resolution but was relatively insensitive. Perbenzoylation has good resolution and good sensitivity, but turns out to be too time consuming. The entire process took 30 hours, including reduction of the anomer and complete perbenzoylation, which was greatly compared to the BAB method, which only took 2 hours. Moreover, the BAB derivatives were stable for several weeks at 4 ° C., but the PMP and AMAC derivatives were much more labile.

Isolation of BAB-labeled (Glc) ₄ from other oligosaccharides found in urine and plasma analyzed a large number of samples from children of various ages known to have no disease and proper selection of elution solvents and flow rates. By (controls). The specificity of the method was substantially ensured by the absence of known interference signals at the retention time of (Glc) ₄ , determined by analyzing fractions from patient samples selected by ESI-MS / MS. An example of normal urine chromatogram showing separation of (Glc) ₄ from other oligosaccharides is shown in FIG. 1 (Panel A) . For comparison, urine from GSD-II patients showing a much larger signal for (Glc) ₄ was included (FIG. 1, Panel B) . Other urine oligosaccharides identified are labeled in FIG . 1 (Panel A and Panel B) . It is noteworthy that the relatively low glucose signal in the urine of patients is consistent with the hypoglycemia phenotype of Pompe disease (Chen, YT and Burchell, A., (1995) in The Metabolic and Molecular Bases of Inherited Disease, 7 ^th Edition, Volume 2 (Scriver, CR, Beaudet, AL, Sly, WS, and Valle, D. Eds), pp. 935-965, McGraw-Hill, New YORK). Comparison of plasma (Glc) ₄ levels was also performed in normal controls (FIG. 2, Panel A) and GSD-II patients (FIG. 2, Panel B) . Large glucose signals were excluded for brevity.

It should be borne in mind that the BAB method cannot separate (Glc) ₄ from maltotetraose (M ₄ ). We have demonstrated that M ₄ is absent in selected controls and patients by analysis of PMP derivatives, which allows for full separation of M ₄ from (Glc) ₄ , as shown in FIG . 3 . Based on this method, the M ₄ content in urine was estimated to be 1 μg / ml or less, which is below the detection limit of the BAB method and is therefore considered to be negligible.

Example 4

(Glc) ₄ Sensitivity and Specificity of the HPLC Method for.

The absolute sensitivity of the method for (Glc) ₄ , defined as having a signal-to-noise ratio of 3 or greater, was 3 ng (4.5 pmol) for a single HPLC injection. For urine samples, the detection limit was 1.2 μg / ml based on 50 μl injection from 1 ml total sample volume, and for plasma, the detection limit was based on injection of 100 μl total from 150 μl total. 0.02 μg / ml. This sensitivity was above the appropriate level, based on normal control values (see Table 1).

TABLE 1

In urine and plasma of normal controls (Glc) ₄ density ^One

Urine (mmol / mol creatine)

Age (age) n Average SD ² Max. ³ <11-55-1010-20> 20 2020181220 8.93.620.90.4 8.23.82.11.00.3 26.912.55.73.81.0

Plasma (µg / mL)

Age (age) n Average SD ² Max. ³ <11-55-1010-20> 20 2020131112 0.20.150.150.130.08 0.260.100.100.100.06 0.370.350.360.320.18

¹ Five age groups were tested.

² Standard Deviation

³ Maximum (Glc) ₄ concentration measured in the corresponding age group

Example 5

(Glc) ₄ Accuracy and Precision of HPLC Analysis for

Internal standards (C5) were introduced to account for losses occurring during sample preparation and analysis. Applied (Glc) urine standard curve of water ₄ (Glc) of the peak area ratio of the C5 ₄ concentration was linear up to 15 ㎍ / ml, which was corresponding to 300 ㎍ / ml concentration in the urine sample. The plasma standard curve was linear down to 1 μg / ml, corresponding to a 7.5 μg / ml concentration in the plasma sample. The r ² value for linear regression was> 0.999. The accuracy and precision of the method was within acceptable limits for clinical analysis, according to the duplicate analysis of calibrators ( Table 2 ) . Reproducibility of the method was determined by analyzing control samples of equal quality at weekly intervals. As shown in Table 2, the results were within 10% tolerance for urine and plasma control samples.

TABLE 2

Interday (Glc) ₄ Analysis accuracy and precision

According to compensators Urine (n = 4) Plasma (n = 4) True value (μg) Mean (μg) cv (%) error(%) True value (μg) Mean (μg) cv (%) error(%) 0.51.02.57.515 0.531.022.577.3815.02 9.347.134.522.421.91 5.21.482.63-1.590.13 0.050.10.20.51.0 0.050.110.210.501.01 8.767.766.583.552.93 -2.566.985.55-0.151.38

In accordance with Quality Controls Urine (μg) Plasma (μg) Low QC (n = 12) High QC (n = 12) Low QC (n = 4) High QC (n = 10) Average0.83 cv (%) 8.3 Average5.82 cv (%) 5.9 Average0.076 cv (%) 10.1 Average0.65 cv (%) 9.7

cv (%): 100% * standard deviation / mean

Error (%): 100% * error / average

Example 6

Identification of oligosaccharides isolated by HPLC by ESI / MS / MS

If HPLC chromatography separation was deemed appropriate, ESI-MS was used to confirm the presence of (Glc) ₄ in selected patient urine samples. Fractions corresponding to (Glc) ₄ , collected during HPLC analysis of patient and control samples, were analyzed by ESI-MS. Most were found to be homogeneous for tetraglucose, as determined by the ionic mass m / z 866, which corresponds to the sodium adduct of BAB-labeled glucose tetramers. During the method development, the amount of (Glc) ₄ in some infant control urine samples was higher than expected when analyzed by HPLC. Analysis of the (Glc) ₄ fraction by ESI-MS showed that it was co-eluted with the compound of m / z 688. Using ESI-MS / MS analysis, this compound was found to be the sodium adduct of the deoxyhexose-hexose-hexose PAB derivative. The assumption that these compounds will be 2'-fucosyl-lactose, a component of human breast milk (Chaturvedi, P, et al. (1997) Anal. Biochem. 251, 89-97), HPLC of authentic specimens And by ESI-MS / MS analysis. Next, the HPLC method was suitably modified to separate this compound from (Glc) ₄ . This underscores the value of mass spectrometry in the development of clinical HPLC assays that rely on non-molecularly-specific detectors.

ESI-MS / MS is also used to differentiate (Glc) ₄ from isomer maltotetraose (M ₄ ), since these compounds are not separated by HPLC under these analytical conditions. Collision-induced dissociation (CID) results of Na ⁺ adducts of (Glc) ₄ and M ₄ yielded the fragmentation pattern shown in FIG. 4 . Ions at m / z 704 and 542 are generated by successive loss of glucose residues from the non-reduction-terminus bearing sodium cations on PAB-modified glucose residues, while ions at m / z 509 Resulting from the loss of PAB-glucose residues bearing sodium cations on the non-reducing-terminus. The average (± 2 standard deviation (SD)) intensity ratio of fragment m / z 509 to m / z 542 was determined to be 1.57 (± 0.11) in the (Glc) ₄ spectrum (FIG. 4, Panel A) and the M4 spectrum It was determined to be 0.65 (± 0.05) in (FIG. 4, Panel B) . The error in ratios was 3.6% for (Glc) ₄ and 4.3% for M4 for 7 replicate analyzes performed over 3 weeks. These data suggest that loss of residues from the reduction-terminus in (Glc) ₄ is preferred over loss of residues from the non-reduction-terminus, whereas vice versa for M ₄ . The ratio of m / z 509 to m / z 542 fragment ions from the urine hexose tetramer in six other patients was 1.64 ± 0.44 (mean ± 2), comparable to that of the (Glc) ₄ standard. , Tetramer is actually primarily (Glc) ₄ . An example is shown in FIG. 4 (Panel C) .

ESI-MS / MS is further used to identify the larger oligosaccharides observed in the urine of some patients with GSD-II. An example of an ESI-MS analysis for total BAB-derivatized urine from such patients is shown in FIG. 5 . The ions at m / z 866, 1028, 1190 and 1352 correspond to the sodium adducts of hexose oligomers having 4, 5, 6 and 7 units, respectively. Signals for these ions were much lower or absent in control urine samples, suggesting that they are all derived from glycogen. Analysis of these adducts using ESI / MS / MS has identified product ions with the same masses as those derived from M ₅ , M ₆ and M ₇ standards, confirming that they are hexose oligomers. . However, for (Glc) ₄ and M ₄ , there were differences in the strength of certain ions, which are summarized in Table 3 . The main differences between urine hexose oligomers and M ₅ , M ₆ and M ₇ were m / z 509 for m / z 542, m for m / z 671 and m / z 866 for m / z 704, respectively. / z 833 ratios. These data indicate that in urine hexose oligomers, loss from the reduction-terminus is preferred, since product ions from this fragmentation pathway have a higher strength compared to product ions derived from non-reduction-terminus. Is supported by These results indicate that hexose oligomers in the urine of the patient, as previously reported for glucose oligomers, containing 6 to 8 residues and identified in the urine of patients suffering from GSD-II and GSD-III (Lennartson, et al. (1978) Eur. J. Biochem. 83, 325-334), implying at least one α-1 → 6 linkage.

TABLE 3

Fragment intensity ratios of hexose oligomers and BAB-labeled maltodes standards present in the urine of GSD-II patients analyzed by ESI-MS-MS

oligosaccharide m / z ratio A Strength ratio m / z ratio B Strength ratio M5 hexose pentamer M6 hexose hexamer M7 hexose heptamer 671/866 671/866833/1028833/1028995/1190995/1190 0.781.70.693.00.651.2 671/704671/704833/866833/866995/1028995/1028 0.340.960.501.71.01.94

Loss of BAB-labeled hexose at the reducing end produces m / z 671, 833, and 995 fragment ions of hexose pentamers, hexamers, and heptamers, respectively. Of these ions:

A. by losing one residue from the non-reducing end (m / z 866, 1028, and 1190, respectively),

B. By losing two residues from the non-reducing end (m / z 704, 866, and 1028, respectively) the ratios for those produced were shown.

These ratios are based on the average of three replicate infusions for maltomeric standards and the average of two different patient samples for urine oligomers.

Example 7

Urine and Plasma (Glc) ₄ Concentration

Urine and plasma (Glc) ₄ concentrations of normal controls, separated by age range, are summarized in Table 1. An inverse relationship was found in which (Glc) ₄ secretion decreased with increasing age, which was more quantitatively distinct in urine than in plasma. The concentrations of (Glc) ₄ in urine and plasma of patients suffering from GSD-II were also shown to be age-dependent, as shown in Tables 4 and 5.

TABLE 4

Urine in Glucose Accumulation Patients (Glc) ₄ Levels

condition Age (age) (Glc) ₄ (mmol / mol Cr ¹ ) (Glc) ₄ ² (normal range) (mmol / mol Cr ¹ ) GSD ⅡGSD ⅡGSD ⅡGSD ⅡGSD ⅡGSD Ⅱ 0.10.20.50.50.80.9 34.445.545.631.517.633.1 8.9 ± 8.2 GSD ⅡGSD ⅡGSD ⅡGSD ⅡGSD Ⅱ 2.53.04.04.05.5 54.752.227.292.873.4 3.6 ± 3.8 GSD II 11 31.0 2.0 ± 2.1 GSD ⅡGSD ⅡGSD ⅡGSD ⅡGSD ⅡGSD Ⅱ 203140454561 33.025.02.24.88.86.5 0.4 ± 0.3 GSD Ia GSD Ia 26 4.84.8 3.6 ± 3.8 GSD Ib 19 0.8 2.0 ± 2.1 GSD IIIaGSD IIIGSD IIIb 459 18.297.623.9 3.6 ± 3.8 GSD IIIaGSD IIIGSD IIIb 283946 4.81.82.1 0.4 ± 0.3

¹ Cr = Creatine

² Urine (Glc) ₄ level (mean ± standard deviation) of normal subjects in the corresponding age groups.

TABLE 5

Plasma (Glc) in Patients with Glucose Accumulation Disease Type II ₄ Levels

age (Glc) ₄ (μg / mL) (Glc) ₄ ¹ (normal range) (μg / mL) 0.10.20.50.80.50.9 0.71.191.134.82.242.0 0.2 ± 0.26 2.52.53.04.04.0 0.892.161.470.512.15 0.15 ± 0.1 5.5 0.37 0.15 ± 0.1 20314044454561 0.870.670.120.660.190.170.08 0.08 ± 0.06

¹ Plasma (Glc) ₄ level (mean ± standard deviation) of normal subjects in the corresponding age groups.

It has previously been reported that the secretion of urine (Glc) ₄ is affected by diet, fasting status, and physical activity (Walker, GJ and Whelan, WJ ( 1960), Biochem. J. 76, 257-263). In Table 1 urine (Glc) ₄ levels were normalized to urine creatine concentrations. No attempt was made to control diet and physical activity factors during sample collection. However, results from 21 patients with GSD-II (infant, child, and adult forms) showed that concentrations of (Glc) ₄ in both plasma and urine were at least a factor higher than those of normal controls at that age. Appeared to be higher than "2". Table 4 also shows the concentration of urine (Glc) ₄ for some patients suffering from GSD-I and GSD-III. Patients suffering from GSD-III accumulate glycogen and secrete higher levels of (Glc) ₄ . (Glc) ₄ has been shown in vitro experiments to limit dextrin resulting from α-amylase degradation of glycogen (Walker, GJ and Whelan, WJ (1960), Biochem. J. 76, 257-263, Ugorski, M., et al. (1983) J. Exp. Pathol. 1, 27-38). Intravenous injection of glycogen (Glc) ₄ in Rhesus monkeys has been shown to increase (Kumlien, J., et al. (1988) Clin. Chim. Acta 176 , 39-48). Acid α-glucosidase (GAA) has been reported to degrade glycogen at both 1 → 4 linkage and 1 → 6 linkage sites (branch sites) (Brown, BI et al. (1970) Biochem. 9, 1423-1428) . Thus, glycogen accumulated by deficiency of GAA (GSD-II) and debranching enzyme (de-branching enzyme) may have a similar structure. Increased glycogen release into the circulation, due to breakdown and turnover of glycogen-laden tissues, is expected to increase (Glc) ₄ concentrations in plasma and urine. The concentrations of urine (Glc) ₄ in GSD-I patients are within the control range. This may be due to the fact that in GSD-I, the main storage substance is fat rather than glycogen (Chen, YT and Burchell, A., (1995) in The Metbolic and Molecular Bases of Inherited Disease, 7 ^th Edition, Volume 2 (Scriver, CR, Beaudet, AL, Sly, WS, and Valle, D. Eds), pp. 935-965, McGraw-Hill, New York.

Example 8

Application of HPLC Methods for Clinical Diagnosis

Plasma (Glc) ₄ levels of GSD-II patients were first reported herein. All infantile and juvenile onset patients had significantly higher levels, while two patients with adult-onset GSD-II were within normal range. The concentration of urine (Glc) ₄ measured for GSD-II patients in this experiment was 19 to 821 nmol / mg creatine (2.2 to 92.8 mmol / mol creatine), which was reported by Oberholzer and Sewell. Comparable to the numerical range from GSD-II patients ((1990) Clin. Chem. 36, 1381). The values reported by Peelen et al. (78-324 mmol / mol creatine) are much higher ((1994) Clin. Chem. 40, 914-921). Lundblad and co-workers have reported (Glc) ₄ concentrations as a secretion rate of mg / 24 hours ((1976) Biomed. Mass SPectrom. 3, 51-54). This may provide the most accurate data on the (Glc) ₄ concentration status, but 24-hour urine sample collection is not practical for clinical diagnostic settings. Based on the results obtained in this study, spot urine samples (with known creatine levels) from patients with high clinical probabilities or risks for Pompe disease were compared with appropriate control age ranges. If used, it is sufficient to make a presumptive diagnosis within 24 hours of sample receipt. Next, muscle biopsies or dermal fibroblasts may be recommended to assure disease diagnosis by traditional enzymatics.

Example 9

(Glc) as a live marker ₄ Use of

Study subjects.

Study for Type I / II clinical trials of enzyme replacement therapy in three infants who have been shown to have infantile GSD-II with reduced activity below 1% of normal GAA activity in skin fibroblasts and / or muscles Was adopted. The enzyme source was rhGAA purified from the culture medium of recombinant human GAA (rhGAA) secreting CHO cells. The study was approved by an institutional review board, with written parental consent. Detailed patient characteristics and clinical evaluation of cardiac, lung, neurological and motor functions are described in a paper reporting clinical results (Amalfitano A, et al. (2001) Genet. Med. 3 : 132). These three patients had different clinical presentations in terms of severity at the start of treatment. Patient 1 was treated at 4 months of age (Pt1) and had massive cardiomegaly, severe motor delay, and feeding difficulty, accompanied by failure to thrive. Was in the most advanced state. Pt2 started treatment at 3 months of age and showed extensive cardiac hypertrophy, moderate exercise delay and difficulty eating. Pt3 was in the early stages of the disease when treatment began at two and a half months of age. Pt3 had a significant motor delay, but no cardiac hypertrophy symptoms.

(Glc) ₄ Measure.

Plasma and 6 hour urine were collected every two weeks before and during treatment. Both urine and plasma samples were frozen at −20 ° C. prior to testing. Urine and plasma (Glc) ₄ levels were measured by HPLC using BAB as a derivative as described in Example 2 above. For qualitative analysis, C5 was added to each test sample as an internal standard, and two control samples ((Glc) ₄ low and high) were measured daily.

Monitoring of disease progression and response to treatment using HPLC methods.

Urine (Glc) ₄ levels for three patients before and during treatment are shown in FIG. 6 . Levels in plasma (Glc) ₄ are shown in FIG. 7 . Plasma (Glc) ₄ levels correlated with clinical severity at the start of treatment. Pt1 at the most advanced stage of the disease exhibited the highest levels of (Glc) ₄ in plasma and urine, while Pt2, which was moderately ill, exhibited moderately elevated levels of (Glc) ₄ content. It was. Pt3, which showed the most mild signs of disease, showed an increase in plasma (Glc) ₄ only and was normal in urine. As a biomarker for Pompe disease, the plasma (Glc) ₄ measurement appears to be more sensitive than the urine measurement. This can be seen from the fact that the degree of elevation of plasma (Glc) ₄ is higher than that of urine (Glc) ₄ levels and that Pt3 has normal urine levels.

During the initial two to three months of treatment, (Glc) ₄ levels in urine and plasma were reduced for all three patients (FIGS. 6 and 7) . In addition, the decrease was more pronounced in plasma than in urine. However, (Glc) ₄ levels in Pt1 and Pt2 subsequently rose, accompanied by clinical decline and production of antibodies to rhGAA. In an attempt to remove the antibody and induce resistance to plasmaphresis, IVIG, cytoxan and daily enzyme injection for 10 days resulted in temporary clinical improvement, but then again clinical decline Accompanied by Pt1, a second immuno-resistant therapy was needed. There was a correlation between the levels of (Glc) ₄ and the clinical course and response to immuno-resistant therapies for Pt1 (FIGS. 6 and 7) . Similar correlations between clinical course and response to immuno-resistant therapies were observed in Pt2. Pt3, which did not produce anti-rhGAA antibodies, maintained normal cardiac, nerve and motor functions. (Glc) ₄ levels for Pt3 normalized during the first two months of treatment and remained nearly normal after that. The correlation between clinical course and (Glc) ₄ levels in all three patients was once again more pronounced in plasma than in urine. The results strongly suggest that (Glc) ₄ levels, in particular plasma (Glc) ₄ levels, are indicative of the clinical state of the disease in Pompe patients. Measurement of (Glc) _{4 in} the blood appears to provide a non-invasive way to analyze the overall disease state and therapeutic response to treatment in Pompe disease, and thus muscle biopsy may be avoided.

Example 10

(Glc) by Tandem Mass Spectrometry ₄ Synthesis of Stable Isotope-labeled Internal Standards for Analysis

A stable, isotope-labeled internal standard was synthesized using the method of Tonozuka and co-workers (Tonozuka at al., (1994) Carbohydrate Research 261 ; 157; Tonozuka et al., (1996) J. Appli. Glycosci. 43:95 ). In this method, pullulan, a polymer of trisaccharide panose (Glcα1-6Glcα1-4Glc), was degraded by α-amylase, TVA II in the presence of [U- ¹³ C] glucose. . TVA II catalyzes the transglycosilation of panoose products and adds [U- ¹³ C] glucose to both α1-4 and α1-6 linkages, thereby reducing glucose with labeled residues at the reducing end Form tetramers.

100 mg pullulan and 100 mg [U- ¹³ C ₆ ] glucose were dissolved in 2 ml of 50 mM sodium citrate at pH 6.9 and mixed with 100 μl of NaHPO ₄ buffer at pH 6.0 containing 8 mg TVA II. Next, incubated for 5.5 hours at 40 ℃. The mixture was cooled on ice and centrifuged at 2000 g for 2 hours through an Amicon Centrifree 30 kDa molecular weight cut-off filter. The filtrate was fractionated on a gel filtration column (170 × 15 cm) filled with Toyopearl HW-40S (Sulpeco, Bellefonte, PA) of 30 μm particle size and eluted with dH ₂ O at a flow rate of 0.5 ml / min. Fractionated fractions are mixed with 15 mM ammonium acetic acid in 65:35 acetonitrile: H ₂ O and 3.5 kV capillary, using acetonitrile: H ₂ O (1: 1, v / v) as mobile phase. And electrospray ionization-mass spectrometry (ESI-MS) using a cone setting of 31 V. Fractions containing [ ¹³ C ₆ ] -labeled hexose tetramers were collected and dried under vacuum at 40 ° C. for 6 hours using Centrivap (Labconco). [ ¹³ C ₆ ] -labeled hexose tetramers were reconstituted in H ₂ O. The combined concentration of [ ¹³ C ₆ ] -labeled hexose tetramers is based on the strength of [M + Na] ⁺ of BAB-derivatized internal standard (m / z 872), unlabeled BAB-derivatized By comparing with that of the Glc ₄ standard (m / z 866), it was determined using ESI-MS. The purity of the internal standard (IS) was determined by analyzing BAB derivatives using HPLC with an ultraviolet detector. Derivatized oligosaccharides were 0.05 mol / L ammonium acetic acid: acetonitrile in 5:22:73 (v / v / v) for 36 minutes on a YMC-Pack Pro C ₁₈ column (250 × 4.6 mm ID, 5 μm): from _{H 2 O 5:60:35 (v / v} / v) 0.05 mol / L ammonium acetate, of: was isolated by gradient elution up in _{H 2 O (v / v)} : acetonitrile. Oligosaccharides were identified by comparing the retention times against true standards. In addition, fractions were collected from HPLC separations and dried under nitrogen gas to remove [M + Na] ⁺ ions by ESI-MS and ESI-MS / MS under the same conditions as for plasma samples described below. Analyzed.

HPLC analysis of the tetrahexose fraction, separated from the reaction mixture by gel filtration, illustrates the presence of numerous components (see FIG. 8 ). Fractions collected from HPLC analysis were analyzed using ESI-MS and ESI-MS / MS. Four components with m / z values and fractionation patterns expected for BAB derivatized [ ¹³ C ₆ ] tetrahexose were identified (peaks 1-4 in FIG. 8 ). One of these components (peak 4 in FIG. 8 ) is [ ¹³ C ₆ ] Glc ₄ derived from its retention time and the ratio of sodiated B ₃ and Y ₂ fragment ions (m / z 509 and 548, respectively). It was confirmed. As described above (see Example 6: see also An et al., (2000) Anal. Biochem. 287 : 136), the ratio of these two product ions to Glc ₄ and maltotetraose with both α1-4 linkages There was a difference. The ratio of 509 to m / z 548 (mean ± 2SD) was found to be 1.33 ± 0.24 (n = 10) for this internal standard component. For the Glc ₄ standard, the ratio (mean ± 2SD) of m / z 509 to 542 (unlabeled equivalent to m / z 548) was 1.41 ± 0.09 (n = 5). Peak 2 was the major isomer present and was believed to be IMIM identified by Tonozuka et al as one of the major products of the transglycosylation reaction. The ratio of 509 to m / z 548 for this isomer was 0.34 ± 0.10 (n = 10), which was similar to that of maltotetraose, 0.48 ± 0.16 (n = 5). The ratio to peak 1 was 1.34 ± 0.32 (n = 8). The structural identity of peaks 1 and 3 is unknown and peak 3 was a mixture of tetramers and pentamers. Panoz (peak 5) and glucose (peak 6) were also present in the preparation. The three major isomers, peaks 1, 2 and 4 together constituted 84% of the internal standard mixture, as determined by HPLC and MS analyzes. The ratio of peak 1: peak 2: peak 4: was 0.2: 1.7: 1.0.

Example 11

Plasma and Urine Glcs ₄ Method for analyzing tandem mass spectrometry

Urine and plasma samples from control and patient were used, stored at -70 ° C or -20 ° C for up to 1 year. Normal human serum (# 1101) was obtained from Biocell Laboratories (Rancho Dominguez, CA). Urine, urine plaques and plasma samples were derivatized with BAB in the manner described above.

Urine: 50 μl of urine was vortex mixed with 25 μl 100 μmol / L internal standard for 10 seconds and with 140 μl of reagent (containing 149 mmol / L BAB, 400 mmol / L NaBH ₃ CN and 6% glacial acetic acid in methanol). Derivatization at 80 ° C. for 45 minutes. Urine samples from patients suffering from GSD II needed to be diluted prior to analysis by mixing 200 μl urine with 1.8 mL dH ₂ O.

Urine plaque: Urine was centrifuged at 14000 rpm for 5 minutes and the supernatant was transferred to a clean tube. 30 μl of the supernatant was spotted onto cotton linter paper (grade 903, Schleicher & Schuell, Keene, NH) and dried overnight at room temperature. The remaining urine was stored at -70 ° C. 2 × 1/4 inch urine plaques were extracted by shaking in 300 μl dH ₂ O and shaking at room temperature for 1 hour. 100 μl of the extract was mixed with 50 μl, 2 μM internal standard, dried under nitrogen and then reconstituted in 20 μl dH ₂ 0 to derivatize as described above.

Plasma and Serum: 100 μl of plasma or serum and 50 μl, 2 μmol / L internal standard were vortex mixed with 500 μl methanol and centrifuged at 14000 g for 5 minutes. The supernatant was dried under N ₂ , reconstituted in 20 μl dH ₂ O and then derivatized for 45 minutes at 80 ° C. using 100 μl reagent (containing 400 mM BAB, 2.0 mol / L NaBH ₃ CN and 7.5% glacial acetic acid in methanol). It was made.

Derivatized urine, urine plaque extracts and plasma samples were purified using solid phase extraction with a C18 cartridge as described above (see Example 2 ; and An et al., Anal. Biochem. 287 : 136). The eluate was dried under 40 ° C., N ₂ , reconstituted in 80:20 methanol: H ₂ O (v / v) and then transferred to a 96-well microtiter plate.

Calibrators: Urine correction groups were prepared using control adult urine to which Glc ₄ standards in the range of 2.5 to 200 μmol / L were added. The urinary plaque group was prepared using dH ₂ O and ranged from 0.1 to 10 μmol / L. Plasma correction groups and quality control samples were prepared using Biocell normal human serum with Glc ₄ standard in the range of 0.1-10 μmol / L.

Mass Spectrometric Analysis and Quantitation: Urine and plasma samples were analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS / MS) using multiple reaction monitoring. (Glc) ₄ and internal standards were detected according to the transition from m / z 866 to m / z 509 and the transition from m / z 872 to m / z 509, respectively. Urine-derivatized oligosaccharide derivatives were injected into an 80:20 methanol: H ₂ O (v / v) mobile phase at a flow rate of 40 μl / min. Plasma and urine plaque samples were subjected to a 2 × 100 mm C18 column (Keystone Scientific Inc.) with a mobile phase of 80:20 methanol: H ₂ O (v / v) at a flow rate of 200 μl / min to concentrate the samples. Analyzes were performed using the same method with additional liquid chromatography steps. The total analysis time was 2.5 minutes for urine samples and 3.0 minutes for plasma and urine plaque samples. A cone voltage of 90 V, a capillary voltage of 3.5 kV, an impact energy of 40 eV and an argon impingement gas pressure of 3.1 × 10 ⁻³ mBar were used. Samples were quantified using an external calibration curve derived by plotting the ratio of MRM signals to the (Glc) ₄ standard relative to the internal standard to the (Glc) ₄ concentration applied.

Creatine Determination: Glc ₄ concentrations in urine and urine plaque extracts were related to the concentration of creatine. Urinary creatine was measured using the picric acid method (Jaffe et al., (1886) Physiol. Chem. 10 : 391), and the corresponding urine and urinary plaque extract samples were published in other literature. It was measured by ESI-MS / MS using a stable isotope dilution method.

Effectiveness of Urine and Plasma Analysis by ESI-MS / MS: Urine and plasma analyzes were validated by repeated analysis of calibrator and quality control (QC) samples. In addition, Glc ₄ concentrations in patient and control samples determined by ESI-MS / MS were compared with the results determined by HPLC-UV. Urine correction curves and QCs were analyzed over a four week period and plasma correction curves and QCs were analyzed over an eight week period.

Urine correction curves and QD interday variation: The urine correction curve is divided into two concentration ranges to quantify control and patient samples where the concentration of Glc ₄ can vary by one or two orders of magnitude. Was split across. The change over time of the slope of the calibration curve was 1.3% for the low concentration range of 2.5 to 70 μM and 2.3% for the high concentration range of 40 to 200 μmol / L (n = 5). The SD coefficient of determination (r ² ) of the mean ± correction curve was (n = 5), 0.998 ± 0.001 and 0.998 ± 0.001 for the low and high concentration ranges, respectively, over four weeks. The accuracy and mean accuracy of the calibration curve over time is shown in Table 6. Intraday and interday precision (cv), determined by repeated analysis of patient samples with mean Glc ₄ concentration of 31.6 mmol / mol creatine, were 2.6% (n = 5) and 5.0% (n = 4) was. For control samples with an average Glc ₄ concentration of 0.4 mmol / mol creatine, the same and two-day precisions were 4.6% (n = 5) and 24.2% (n = 4), respectively.

TABLE 6

Nominal μmol / L Average concentration (mean) μmol / L cv (%) error % Low concentration calibration range (2.5 to 70 μmol / L) 2.55204070 2.44.420.540.869.5 36.015.76.11.80.9 3.513.0-2.3-2.00.8 High concentration calibration range (40 to 200 μmol / L) 4070100150200 40.269.0100.3151.3199.2 2.42.23.02.31.1 -0.41.4-0.3-0.90.4

Changes over time of plasma calibration curves and QCs: Plasma was quantified over 0.1-2.5 μmol / L and 1.0-10 μmol / L, with changes in the calibration curve slope over 8 weeks, 20.3%, respectively. And 20.6% (n = 7). Mean ± SD r ² values were 0.998 ± 0.001 for the low concentration range and 0.994 ± 0.009 for the high concentration range (n = 7). The daily precision and mean accuracy of the calibration curves are shown in Table 7. Plasma QCs were prepared using the same normal human plasma pool used to prepare the correction groups. Small amounts of endogenous Glc ₄ in these plasmas were detected and determined and taken into account in the calculations. Results for two-day and same-day repeated analysis of plasma QCs are shown in Tables 8 and 9, respectively.

TABLE 7

Nominal μmol / L Average concentration (mean) μmol / L cv (%) error % Low concentration calibration range (0.1 to 2.5 μmol / L) 0.10.250.51.02.5 0.080.260.520.982.50 28.08.69.58.00.9 -18.32.53.3-1.9-0.1 High concentration calibration range (1.0 to 10.0 μmol / L) 1.02.55.010.0 1.072.665.6310.10 11.99.011.99.0 7.16.37.16.3

TABLE 8

Two-day analysis Nominal μmol / L Average concentration (mean) μmol / L cv (%) (n = 5) Average error% (n = 5) QC 1QC 2QC 3 0.201.258.0 0.181.308.71 21.57.51.8 -8.54.28.9

TABLE 9

Two-day analysis Nominal μmol / L Average concentration (mean) μmol / L cv (%) (n = 7) Average error% (n = 7) QC 1QC 2QC 3 0.201.258.0 0.241.298.43 36.314.211.2 22.13.35.4

Example 12

Comparison of ESI-MS / MS and HPLC Analyzes

The results of 24 urine samples and 29 plasma samples (patients and controls) analyzed by HPLC methods were compared with the results by the ESI-MS / MS method ( FIGS. 9 and 10 ). For urine samples, y = 0.97x-4.0; S _{y / x} = 6.5 and r ² = 0.82. For plasma samples, y = 0.62x + 0.16; S _{y / x} = 0.31 and r ² = 0.502. Using a Bland-Altman analysis of the data (Bland et al., (1986) Lancet 1 : 307), the consensus range for urine and plasma is (limits of agreement) [mean difference (HPLC-ESI-MS / MS) 2 SD of ± difference] was 3 ± 12.4 and 0.1 ± 0.72, respectively.

Example 13

Glc in liquid and spotted urine samples ₄ Comparison of Analysis

Glc ₄ concentrations were determined from 37 pairs of liquid and spot urine samples. A comparison result of the concentrations is shown in FIG. 11 . y = 0.99x-0.38; S _{y / x} = 5.36 and r ² = 0.954. The limits of agreement for the Bland-Altman analysis [2 SD of Mean Difference (HPLC-ESI-MS / MS) ± Difference] were 0.55 ± 10.4.

Example 14

Investigate what may interfere with the analysis

High cone voltage (90V) was used to optimize the strength of [M + Na] ⁺ ions. At this voltage, some in-source fragmentation occurs, so larger mass hexose oligomers, fragmented, resulting in m / z 866 product ions can interfere with Glc ₄ analysis. have. To investigate this, the degree of phosphorus-source fragmentation of maltopentaose and maltohexaoz at different cone voltages was determined. In addition, the contribution to m / z 866 of heaviose oligomers with greater mass in GSD II patient samples was evaluated. A mass spectrometer was injected with 12 μmol / L maltopentaose and maltohexaose BAB-derivatives in 80:20 methanol: H ₂ O (v / v) using an injection pump (model) at a flow rate of 10 μl / min. The cone voltage was increased from 30 V to 100 V and the relative intensities of m / z 866 and [M + Na] ⁺ were determined. In the case of maltopentaose, the relative intensity of m / z 866 increased with cone voltage, from 2.5% of the intensity of m / z 1028 at 30V to 4.6% at 100V. In the case of maltohexaoz, the relative strength of m / z 866 did not increase with cone voltage. At 90 V, the relative intensities of [M + Na] ⁺ for both standards were comparable (4%). BAB-derivatives of 12 GSD II patient urine samples were analyzed in MS1 mode by scanning between m / z 830 and 1400 at a rate of 100 amu.sec ⁻¹ . The average relative intensities of [M + Na] ⁺ for m / z 866 of hexose pentamer (s), hexose hexamer (s) and hexose heptamer (s) present are 5.2, 2.8 and 3.6, respectively. % Was determined. Thus, the combined contribution of these hexose oligomeric oligosaccharides to the m / z 866 signal was determined to be 0.46%.

Example 15

Neonatal Screening Analysis Using TMS

TMS-based assays were used to screen newborns with Pompe disease using (Glc) ₄ as a live marker. Newborn screening cards containing dry blood spots (typically obtained by heel blood collection) were prepared. The plaques of the disc mothers were removed from the card and placed in a vial containing a solvent for extracting oligosaccharides. An internal standard was added to each vial in a known amount (eg, (Glc) ₄ tetramer in which one of the monomers was substituted with a U- ¹³ C-glucose analog). Oligosaccharides were then derivatized in the sample using butyl-PABA. Derivatized samples were analyzed by TMS as described in Example 11 . Data were collected by computer and analyzed to determine the concentration of (Glc) ₄ in each sample (ie, by comparing the ratio of signals produced by the analyte with the internal standard). Levels above the baseline can be an indicator of Pompe disease.

Optionally, the analyte may contain paramagnetized polystyrene spheres (Dynabeads) chemically conjugated to anti- (Glc) ₄ antibodies. ) And the sample is concentrated before derivatization. Beads are collected by a magnet and the analyte is eluted from the beads using a suitable solvent.

As another optional step, the concentrate of glucose monomer is reduced in the sample prior to TMS analysis. In one protocol, glucose oxidase is added to the sample prior to derivatization. In another alternative protocol, the derivatized glucose monomer is separated by a liquid chromatography step (reverse phase) prior to the TMS step.

The above examples are intended to illustrate the invention and should not be construed as limiting the invention. The invention is described in the following claims, with equivalents of the claims to be included therein.

Claims (54)

A method for screening glycogen accumulation bottles in a subject, the method comprising determining a concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from a subject, and comparing the concentration to a reference value, wherein the biological And when (Glc) ₄ in the sample is detected at a value higher than the reference value, the subject is confirmed to have glycogen accumulation disease. The structure of claim 1, wherein (Glc) ₄ is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. Characterized by having a. The method of claim 1, wherein the concentration of (Glc) ₄ is determined using a quantitative method. The method of claim 3, wherein (Glc) ₄ is selected from the group consisting of tandem mass spectrometry, mass spectrometry, liquid chromatography, and immunopurification. Characterized in that quantified by the selected method. The method of claim 1, wherein the concentration of (Glc) ₄ is determined using a semi-quantitative method. The method of claim 1, wherein the glycogen accumulating disease is selected from the group consisting of Pompe disease (glucogen accumulating disease type II), glycogen accumulating disease type III, and glycogen accumulating disease type IV. The method of claim 1, wherein the subject is a human subject. 8. The method of claim 7, wherein said human subject is a newborn subject. The method of claim 1, wherein the biological sample is a body fluid sample. 10. The method of claim 9, wherein the bodily fluid sample is selected from the group consisting of blood, plasma, serum, urine, saliva and amniotic fluid. The method of claim 10, wherein the bodily fluid sample is a newborn blood sample. 12. The method of claim 11, wherein said newborn blood sample is a dried blood spot. 10. The method of claim 9, wherein the body fluid sample is a dry urine sample. The method of claim 1, wherein the biological sample is a cell or tissue sample. The method of claim 1, wherein the reference value is a predetermined value. The method of claim 1, wherein the reference value is based on concentrations of (Glc) ₄ found in a corresponding population of subjects. The method of claim 16, wherein the corresponding population of subjects is a population of subjects not suffering from disease. The method of claim 1, further comprising performing additional diagnostic tests on the subject identified as having glycogen accumulation disease. A method for screening Pompe disease (Glycogen accumulation bottle type II) in a subject, the method comprising the steps of: determining the concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from the subject, and comparing the concentration to a reference value Wherein the subject is identified as having Pompe disease if (Glc) ₄ in the biological sample is detected at a value higher than a reference value. The structure of claim 19, wherein (Glc) ₄ is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. Characterized by having a. The method of claim 19, wherein the concentration of (Glc) ₄ is determined using a quantitative method. The method of claim 20, wherein (Glc) ₄ is quantified by tandem mass spectrometry. The method of claim 22, wherein the oligosaccharides in the biological sample are derivatized with butyl-para-aminobenzoic acid prior to tandem mass spectrometry. 23. The method of claim 22, wherein the quantitation by tandem mass spectrometry is normalized using [U- ¹³ C] glucose labeled hexose tetramer as internal standard. The structure of claim 24, wherein the internal standard has a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. And a [U- ¹³ C] labeled hexose tetramer with a. The method of claim 19, wherein the concentration of (Glc) ₄ is determined using a quasi-quantitative method. 20. The method of claim 19, wherein the reference value is a predetermined value. The method of claim 27, wherein the predetermined reference value is based on concentrations of (Glc) ₄ found in the corresponding population of subjects. The method of claim 28, wherein the corresponding population of subjects is a population of subjects that do not suffer from the disease. 20. The method of claim 19, further comprising performing additional diagnostic tests on a subject identified as having Pompe disease. In a method for screening Pompe disease (Glycogen accumulation disease type II) in a newborn subject, the step of determining the concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from the newborn subject, wherein (Glc) ₄ is having a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc; And comparing the concentration with a reference value, wherein if the (Glc) ₄ in the biological sample is detected at a value higher than the reference value, the newborn subject is identified as having Pompe disease. A method of monitoring the clinical conditions of a subject suffering from Pompe disease (Glucose Accumulation Disease Type II), the method comprising the steps of: determining the concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from the subject, and the concentration Comparing the to a reference value, wherein if (Glc) ₄ in the biological sample is detected at a value higher than the reference value, the indicator is indicative of the clinical condition of the subject. The structure of claim 32, wherein (Glc) ₄ is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc Characterized by having a. 33. The method of claim 32, wherein the subject is a subject undergoing treatment for Pompe disease. 35. The method of claim 34, wherein said treatment is selected from the group consisting of enzyme replacement therapy, gene therapy, and diet. The method of claim 34, wherein said monitoring is performed to determine to initiate or resume treatment of said subject for Pompe disease. A method of assessing the effectiveness of a therapeutic regimen for a patient suffering from Pompe disease (Glycoaccumulative Disease Type II), comprising the steps of: determining the concentration of hexose tetrasaccharide (Glc) ₄ in a biological sample obtained from a subject, and Comparing the concentration to a reference value, wherein if (Glc) ₄ in the biological sample is detected at a value higher than the reference value, the method is indicative of the effectiveness of the therapeutic regimen for the subject, The structure of claim 38, wherein (Glc) ₄ is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc Characterized by having a. A method of screening Pompe disease (Glycogen accumulation disease type II) in a newborn subject, comprising the steps of: determining the concentration of hexose tetrasaccharide (Glc) ₄ in a dry platelet sample obtained from a newborn subject by tandem mass spectrometry and And comparing the concentration with a reference value, wherein if the (Glc) ₄ in the sample is detected at a value higher than the reference value, the newborn subject is identified as having Pompe disease. The structure of claim 39, wherein (Glc) ₄ is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. Characterized by having a. 40. The method of claim 39, wherein the quantitation by tandem mass spectrometry is standardized using [U- ¹³ C] glucose labeled hexoose tetramer as internal standard. The structure of claim 41, wherein the internal standard has a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. And a [U- ¹³ C] labeled hexose tetramer with a. A method for determining the concentration of oligosaccharides in a biological sample, comprising determining the concentration of hexose tetrasaccharide (Glc) _{4 in} a biological sample obtained from a subject by tandem mass spectrometry. The structure of claim 43, wherein (Glc) ₄ is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc Characterized by having a. The method of claim 43, wherein the oligosaccharides in the biological sample are derivatized with butyl para-aminobenzoic acid prior to tandem mass spectrometry. 44. The method of claim 43, further comprising a concentration step prior to the quantification step. 47. The method of claim 46, wherein said enriching step comprises immunocrystallization. The method of claim 43, wherein the biological sample is selected from the group consisting of blood, plasma, serum, urine, saliva, and amniotic fluid. The method of claim 43, wherein the biological sample is selected from the group consisting of blood, plasma and serum. 44. The method of claim 43, wherein said biological sample is a neonatal blood sample. 44. The method of claim 43, wherein said biological sample is a neonatal urine sample. 44. The method of claim 43, further comprising reducing the concentration of glucose in the biological sample prior to the quantifying step. 44. The method of claim 43, wherein the quantitation by tandem mass spectrometry is normalized using [U- ¹³ C] glucose labeled hexose tetramer as internal standard. The structure of claim 52, wherein the internal standard is a structure of α-D-Glc (1 → 6) -α-D-Glc (1 → 4) -α-D-Glc (1 → 4) -D-Glc. And a [U- ¹³ C] labeled hexose tetramer with a.